Dicoumarol potentiates cisplatin-induced apoptosis mediated by c-Jun N-terminal kinase in p53 wild-type urogenital cancer cell lines

3-3'-Methylene-bis [4-hydroxycoumarin] (dicoumarol), an inhibitor of NADPH:quinone oxidoreductase 1, has been reported to possess potential antineoplastic effects and the ability to abrogate p53 protein. In the present study, we investigated the cytotoxic effects of dicoumarol in combination with cisplatin (CDDP), using four bladder (RT112, 253J, J82 and UMUC3) and two prostate (LNCap and PC3) cancer cell lines. Single treatment with 100 microM dicoumarol suppressed cell proliferation but did not induce apoptosis at 24 h in all cell lines examined. On the other hand, pretreatment with dicoumarol enhanced cytotoxicity of CDDP in three cell lines with wild type of p53 (RT112, 253J and LNCap), but not in three other cell lines with mutant p53 or in RT112 stable transfectants with a dominant-negative mutant of p53. In RT112 and LNCap, CDDP induced p53 and p21 expression, while pretreatment of dicoumarol suppressed induction of p53/p21 and resulted in sequential activation of c-Jun N-terminal kinase (JNK) in a time-dependent manner. Furthermore, inhibition of JNK, using SP600125, completely suppressed activity of caspases and poly-(ADP-ribose) polymerase cleavage, leading to suppression of enhancement of CDDP-mediated apoptosis by dicoumarol. These results suggested that dicoumarol could enhance cytotoxicity of CDDP in urogenital cancer cells with wild-type p53 through the p53/p21/JNK pathways.     

Antimetastatic gene expression profiles mediated by retinoic acid receptor beta 2 in MDA-MB-435 breast cancer cells

Background  

The retinoic acid receptor beta 2 (RARβ2) gene modulates proliferation and survival of cultured human breast cancer cells. Previously we showed that ectopic expression of RARβ2 in a mouse xenograft model prevented metastasis, even in the absence of the ligand, all- trans retinoic acid. We investigated both cultured cells and xenograft tumors in order to delineate the gene expression profiles responsible for an antimetastatic phenotype.     

All-trans retinoic acid inhibits the cell proliferation but enhances the cell invasion through up-regulation of c-met in pancreatic cancer cells

All-trans retinoic acid (ATRA) inhibits proliferation of cancer. However, the effects of ATRA on scattering and invasion of pancreatic cancer cells remain unknown. Also, the effects of ATRA on c-Met expression in pancreatic cancer have never been addressed so far. The effects of ATRA on a pancreatic cancer cell line, Capan-1, were determined by proliferation assay, scattering assay and invasion assay. In addition, the expression of c-Met in pancreatic cancer cell lines treated with ATRA was investigated by real-time PCR and western blotting. The growth-inhibitory effect of ATRA was found when the cells were cultured with 5 microM ATRA for 3 days. In cell scattering assay, ATRA-treated pancreatic cancer cells were found to spread out from their colonies. In invasion assay, cells treated with ATRA invaded the matrigel more than vehicle-treated cells. The expression of c-Met was up-regulated both in the mRNA and protein levels after the treatment of ATRA. The highest expression was found at 48 h after the treatment. ATRA induced scattering and invasion of pancreatic cancer cells, although it inhibited proliferation of those cells. In addition, ATRA also increased the protein level of c-Met. These findings may indicate that the use of retinoic acid as an anti-cancer therapeutic drug needs some additional treatments to control cell invasion or scattering.     

All-trans retinoic acid modulates cancer stem cells of glioblastoma multiforme in an MAPK-dependent manner

Glioblastoma multiforme (GBM), a grade IV glioma, appears to harbor therapy-resistant cancer stem cells (CSCs) that are the major cause of recurrence. All-trans retinoic acid (ATRA), a derivative of retinoid, is capable of differentiating a variety of stem cells, as well as normal neural progenitor cells, and down-regulates expression of the stem cell marker nestin. This study investigated the effects of ATRA on differentiation, proliferation, self-renewal, and signaling pathways of CSCs in GBM. CSCs differentiated into glial and neuronal lineages at low concentrations of ATRA (10 μM). Proliferation and self renewal of neurospheres were reduced following ATRA, although ATRA induced apopotsis at higher (40 μM) concentrations. Analysis of mitogen-activated protein kinase signaling pathways, specifically extracellular signal-regulated kinases (ERK1/2), showed that ATRA-induced alterations in ERK1/2 were associated with regulation of differentiation, proliferation and apoptosis. These results emphasize that low doses of ATRA may have therapeutic potential by differentiating GBM CSCs and rendering them sensitive to targeted therapy.     

All-trans retinoic acid (ATRA)-induced apoptosis is preceded by G1 arrest in human MCF-7 breast cancer cells.

In this study the effects of all-trans retinoic acid (ATRA) on cell cycle and apoptosis of MCF-7 human breast cancer cells were investigated to elucidate the mechanisms underlying the antineoplastic potential of this retinoid in breast cancer. The antiproliferative effect of ATRA was evaluated by DNA content measurements and dual-parameter flow cytometry of bromodeoxyuridine (BrdU) incorporation and of the expression of cell cycle-related proteins (Ki-67 as proliferation marker and statin as quiescence marker) vs DNA content. Apoptosis was also studied by flow cytometry of either DNA content or Annexin V labelling. After 10(-6) M ATRA treatment, the fraction of S-phase cells decreased significantly, and cells accumulated in the G0/G1 range of DNA contents. Dual-parameter flow cytograms showed a decrease in the percentage of Ki-67-labelled cells (after 10 days, only 20% of the cells were still positive for Ki-67 compared with 95% in controls), while the fraction of statin-positive cells increased slightly. From 3 days of treatment onwards, apoptosis was found to occur. These results show that ATRA-induced inhibition of MCF-7 cell growth is related to two mechanisms, i.e. the block of cell proliferation, mostly in a pre-S phase, and the induction of apoptosis. These results should be taken into account when attempting to design treatment programmes that associate ATRA with antineoplastic compounds of different cell cycle specificity.     

All-trans retinoic acid induced the differentiation of human glioma cells

Objective  

To observe the effect of all-trans retinoic acid (ATRA) on inducing human glioma MO59K cells differentiation and further explore the underlying molecular mechanisms.     

Aurora kinase A suppresses metabolic stress-induced autophagic cell death by activating mTOR signaling in breast cancer cells

Aberrant Aur-A signaling is associated with tumor malignant behaviors. However, its involvement in tumor metabolic stress is not fully elucidated. In the present study, prolonged nutrient deprivation was conducted into breast cancer cells to mimic metabolic stress in tumors. In these cells, autophagy was induced, leading to caspase-independent cell death, which was blocked by either targeted knockdown of autophagic gene ATG5 or autophagy inhibitor 3-Methyladenine (3-MA). Aur-A overexpression mediated resistance to autophagic cell death and promoted breast cancer cells survival when exposed to metabolic stress. Moreover, we provided evidence that Aur-A suppressed autophagy in a kinase-dependent manner. Furthermore, we revealed that Aur-A overexpression enhanced the mammalian target of rapamycin (mTOR) activity under metabolic stress by inhibiting glycogen synthase kinase 3β (GSK3β). Inhibition of mTOR activity by rapamycin sensitized Aur-A-overexpressed breast cancer cells to metabolic stress-induced cell death. Consistently, we presented an inverse correlation between Aur-A expression (high) and autophagic levels (low) in clinical breast cancer samples. In conclusion, our data provided a novel insight into the cyto-protective role of Aur-A against metabolic stress by suppressing autophagic cell death, which might help to develop alternative cell death avenues for breast cancer therapy.     

全反式维甲酸对EC9706荷瘤裸鼠抑瘤作用的实验研究

目的：观察全反式维甲酸的体内抗肿瘤作用，探讨其体内抑瘤的作用机制。方法：采用裸鼠EC9706人食管癌细胞实体瘤模型进行体内抑瘤实验研究，应用全反式维甲酸10d后处死裸鼠，观察荷瘤裸鼠的肿瘤生长情况，测定肿瘤生长抑制率；HE染色对肿瘤组织进行细胞形态学观察，TUNEL法检测肿瘤组织中细胞凋亡，免疫组织化学法检测凋亡相关基因bax及bcl-2蛋白的表达情况。结果：全反式维甲酸对裸鼠EC9706人食管癌细胞实体瘤生长具有明显的抑制作用，抑瘤率达60％以上。组织学观察及TUNEL检测结果显示，肿瘤组织中散在凋亡细胞和凋亡小体。免疫组织化学检测结果显示，凋亡相关基因bax蛋白表达增加，bcl-2蛋白表达下降。结论：全反式维甲酸具有较强的体内抗肿瘤作用，作用机制与诱导肿瘤细胞凋亡有关。     

All-trans retinoic acid decreases susceptibility of a gastric cancer cell line to lymphokine-activated killer cytotoxicity.

All-trans retinoic acid (RA) was previously shown to regulate the growth of gastric cancer cells derived from the cell line SC-M1. This study was designed to investigate the effect of RA on the sensitivity of SC-M1 cells to lymphokine-activated killer (LAK) activity. RA at the concentration range of 0.001-10 microM was shown to induce SC-M1 cells to exhibit resistance to LAK activity in a dose-dependent manner. A kinetics study indicated that a significantly increased resistance was detected after 2 days of co-culturing SC-M1 cells with RA and reached a maximum after 6 days of culture. Similar results were obtained from two other cancer cell lines: promyelocytic leukaemia HL-60 and hepatic cancer Hep 3B. A binding assay demonstrated that the binding efficacy between target SC-M1 cells and effector LAK cells was not altered by RA. Flow cytometric analyses revealed that RA exhibited no effect on the expression of cell surface molecules, including HLA class I and class II antigens, intercellular adhesion molecule-1 and -2, and lymphocyte function antigen-3. Cell cycle analysis revealed that culture of SC-M1 cells with RA resulted in an increase in G0/G1 phase and a decrease in S phase, accompanied by a decrease in cyclin A and cyclin B1 mRNA as determined by Northern blot analysis. Additionally, RA was shown to enhance the expression of retinoic acid receptor alpha (RAR alpha) in SC-M1 cells, and to have no effect on the expression of RARbeta or RARgamma. Taken together, these results indicate that RA can significantly increase gastric cancer cells SC-M1 to resist LAK cytotoxicity by means of a cytostatic effect through a mechanism relating to cell cycle regulation. The prevailing ideas, such as a decrease in effector to target cell binding, a reduced MHC class I antigen expression or an altered RARbeta expression, are not involved.     

全反式维甲酸和二甲亚砜诱导人食管癌细胞wtp53基因的表达

目的探讨野生型p53(wtp53)基因表达与食管癌细胞分化相关性。方法采用全反式维甲酸(ATRA)和二甲亚砜(DMSO)诱导人食管癌Eca109细胞分化,应用甲基绿-派洛宁染色、软琼脂集落形成、原位杂交方法分别检测细胞分化和wtp53的基因表达变化。结果ATRA和DMSO作用5d后可以抑制Eca109细胞增殖,诱导细胞分化及降低集落形成率。wtp53基因表达随着Eca109细胞分化而增高。结论ATRA和DMSO可上调Eca109细胞wtp53的表达使其趋向正常细胞分化。     

Cell growth inhibition by all-trans retinoic acid in SKBR-3 breast cancer cells: involvement of protein kinase Calpha and extracellular signal-regulated kinase mitogen-activated protein kinase

All-trans retinoic acid (ATRA), a synthetic derivative of vitamin A, inhibits the growth of breast cancer cells. To elucidate the mechanism by which ATRA causes cell growth inhibition, we examined changes in cell cycle and intracellular signaling pathways, focusing on protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). Using the estrogen receptor-negative, retinoid receptor-positive breast cancer cell line SKRB-3, we found that treatment with ATRA significantly decreased the expression of PKCalpha, as well as reducing ERK MAPK phosphorylation. ATRA treatment leads to dephosphorylation of Rb, and consequently to G(1) arrest. Marked changes in the expression of cyclins (particularly cyclins A and E) were observed in SKBR-3 cells treated with ATRA. Using a series of pharmacological and molecular approaches, we found evidence that ATRA-induced SKBR-3 cell growth inhibition involves the deregulation of the PKCalpha-MAPK pathway. These data suggest that retinoids interfered with signal transduction pathways that are crucial for cell cycle progression, and highlight the complexities of the biological effects of retinoid derivatives.     

All-trans retinoic acid inhibits craniopharyngioma cell growth: study on an explant cell model

To examine factors impacting long-term functional outcomes and psychological sequelae in persons with primary brain tumours (BT) in an Australian community cohort. Participants (n = 106) following definitive treatment for BT in the community were reviewed in rehabilitation clinics to assess impact on participants’ current activity and restriction in participation, using validated questionnaires: Functional Independence Measure (FIM), Perceived Impact Problem Profile (PIPP), Depression Anxiety Stress Scale, Cancer Rehabilitation Evaluation System–Short Form and Cancer Survivor Unmet Needs Measure. Mean age of the participants was 51 years (range 21–77 years), majority were female (56 %) with median time since BT diagnosis 2.1 years and a third (39 %) had high grade tumours. Majority showed good functional recovery (median motor FIM score 75). Over half reported pain (56 %), of which 42 % had headaches. Other impairments included: ataxia (44 %), seizures (43 %); paresis (37 %), cognitive dysfunction (36 %) and visual impairment (35 %). About 20 % reported high levels of depression, compared with only 13 % in an Australian normative sample. Two-third (60 %) participants reported highest impact on the PIPP subscales for psychological wellbeing (scores of >3 on 6-point scale) and participation (45 %). Factors significantly associated with poorer current level of functioning and wellbeing included: younger participants (≤40 years), recent diagnoses, aggressive tumour types and presence of pain. No significant differences in scale scores were found across various treatments (surgery, chemotherapy or radiotherapy) on outcomes used. Rehabilitation for BT survivors is challenging and requires long-term management of psychological sequelae impacting activity and participation. More research into participatory limitation is needed to guide treating clinicians.     

alpha-Tocopheryloxybutyric acid enhances necrotic cell death in breast cancer cells treated with chemotherapy agent

The overexpression of HER-2 receptor contributes to malignant transformation of breast cancer cells. We have reported that alpha-tocopheryloxybutyric acid (TE), non-antioxidative vitamin E ether derivative inhibits the activation of HER-2 receptor. The present study was undertaken to estimate if TE could act as a useful anti-cancer agent against a breast cancer cell overexpressing HER-2 receptor (MDA-MB-453 cell line) in combination with a conventional chemotherapy agent, adriamycin (ADR). TE enhanced cytotoxic effect of ADR against the human breast cancer cell at low doses less than IC(50). The enhancing effect was mainly dependent on the elevation of necrotic-like cell death but not apoptotic cell death. In conjugation with this event, the inactivation of HER-2 receptor in the breast cancer cell was caused by the combination of TE with ADR. These results suggest that TE enhances necrotic-like cell death in the breast cancer cells and that the cell death relates to the inactivation of HER-2 receptor in the breast cancer cells.     

Induction of cell death in antiestrogen resistant human breast cancer cells by the protein kinase CK2 inhibitor DMAT

Protein kinase CK2 is involved in cell proliferation and survival, and found overexpressed in virtually all types of human cancer, including breast cancer. We demonstrate that inhibition of CK2 with 2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole (DMAT), a potent and specific CK2 inhibitor, results in caspase-mediated killing of human breast cancer cells with acquired resistance to antiestrogens, while DMAT fails to kill parental MCF-7 cells. The antiestrogen resistant breast cancer cells express reduced levels of Bcl-2 compared to MCF-7 cells. Reduced Bcl-2 protein level is also found in a tamoxifen resistant human breast tumor grown as a xenograft. We show that re-expression of Bcl-2 partially rescues antiestrogen resistant MCF-7 sublines from DMAT-induced cell death. In summary, our data suggest a novel role of CK2 in antiestrogen resistance.     

Loss of growth inhibitory effects of retinoic acid in human breast cancer cells following long-term exposure to retinoic acid

Although retinoids are known to be inhibitory to breast cancer cell growth, a key remaining question is whether they would remain effective if administered long-term. We describe here the long-term effects of all- trans retinoic acid on two oestrogen-dependent human breast cancer cell lines MCF7 and ZR-75-1. Although both cell lines were growth inhibited by retinoic acid in the short-term in either the absence or the presence of oestradiol, prolonged culture with 1 microM all- trans retinoic acid resulted in the cells acquiring resistance to the growth inhibitory effects of retinoic acid. Time courses showed that oestrogen deprivation of the cell lines resulted in upregulation of the basal non-oestrogen stimulated growth rate such that cells learned to grow at the same rate without as with oestradiol, but the cells remained growth inhibited by retinoic acid throughout. Addition of 1 microM all- trans retinoic acid to steroid deprivation conditions resulted in reproducible loss of growth response to both retinoic acid and oestradiol, although the time courses were separable in that loss of growth response to retinoic acid preceded that of oestradiol. Loss of growth response to retinoic acid did not involve loss of receptors, ER as measured by steroid binding assay or RARalpha as measured by Northern blotting. Function of the receptors was retained in terms of the ability of both oestradiol and retinoic acid to upregulate pS2 gene expression, but there was reduced ability to upregulate transiently transfected ERE- and RRE-linked reporter genes. Despite the accepted role of IGFBP3 in retinoic acid-mediated growth inhibition, progression to retinoic acid resistance occurred irrespective of level of IGFBP3, which remained high in the resistant MCF7 cells. Measurement of AP1 activity showed that the two cell lines had markedly different basal AP1 activities, but that progression to resistance was accompanied in both cases by a lost ability of retinoic acid to reduce AP1 activity. These results warn of potential resistance which could arise on long-term treatment with retinoic acid in a clinical situation and echo the problems of progression to endocrine resistance. It seems that whatever the constraints imposed on growth, these cells have a remarkable ability to escape from growth inhibition. However, the ability of retinoic acid to delay progression to oestrogen resistance is encouraging for endocrine therapy, and the concentration-dependence of retinoic acid resistance suggests that progression is not absolute but could be manipulated by dose.     

c-Jun-NH2-terminal kinase potentiates apoptotic cell death in response to carboplatin in B lymphoma cells

PURPOSE: Exposure to carboplatin (CBDCA) has been demonstrated to result in apoptotic and/or necrotic cell death, but molecular mechanisms underlying CBDCA-induced apoptosis or necrosis remain largely unclear. Here, we examined whether activation of c-Jun NH(2)-terminal kinase (JNK) modulates the mode of cell death induced by CBDCA in CD31 B lymphoma cells. METHODS: The mode of cell death (apoptosis versus necrosis) was investigated by flow cytometry using 7-amino-actinomycin D (7-AAD) and annexin-FITC probes. To evaluate the role of JNK1 in CBDCA-induced cell death, CH31 B lymphoma cells overexpressing dominant-negative form of JNK1 (dnJNK1) or constitutively active form of JNK1 (MKK7-JNK1) were established. Intracellular accumulation of superoxide anion (O(2) (-)) was determined by flow cytometry using the fluorescent probe dihydroethidium (DHE). RESULTS: The CBDCA-induced primary apoptosis and secondary necrosis were abrogated in the dnJNK1-overexpressing CH31 cells, while it was somewhat enhanced in the MKK7-JNK1-overexpressing cells. In contrast, the CBDCA-induced primary necrosis was reduced by MKK7-JNK1, with a concurrent decrease in production of O(2) (-). The superoxide anion scavenger for butylated hydroxyanisol (BHA) partially reduced the CBDCA-induced O(2) (-) production and necrotic, but not apoptotic, death in both wild type and dnJNK1-overexpressing CH31 cells. CONCLUSIONS: Prolonged activation of JNK1 appears to be involved in CBDCA-induced apoptosis with prevention of necrosis induction, and the induction of necrosis appears to correlate with CBDCA-induced O(2) (-) production, which is partially blocked by co-culture with BHA. These observations provide valuable information for understanding molecular mechanisms underlying CBDCA-induced cell death, and hopefully for the design of novel treatment modalities for patients with tumors.     

Retinoic acid attenuates promyelocytic leukemia protein-induced cell death in breast cancer cells by activation of the ubiquitin-proteasome pathway

All-trans-retinoic acid and the tumor suppressor promyelocytic leukemia protein (PML) are potent regulators of the growth of cancer cells. This study investigates the individual and combined effects of PML, when overexpressed by the recombinant PML adenovirus, and all-trans-retinoic acid on the proliferation of human estrogen-receptor negative SKBR-3 and estrogen-receptor positive MCF-7 breast cancer cell lines. All-trans-retinoic acid caused a significant degree of cell death in SKBR-3 cells and MCF-7 cells, and PML elicited a similar incidence of or slightly more cell death in MCF-7 cells. Dual-treated cells displayed significantly less cell death than did single-treated cells in the same cell line. We concluded that PML and all-trans-retinoic acid cause cell death by different pathways: PML activates ERK1/2, p38 MAPK, and p21; arrests the cell cycle; and later causes cell death; and all-trans-retinoic acid activates proteasome function, caspase cleavage, and apoptosis. The combined use of all-trans-retinoic acid and PML gene therapy may not be the best treatment for patients with cancer, because the ubiquitinylation of PML and its subsequent proteasome-dependent degradation by retinoic acids occur before overexpressed PML exhibits tumor-suppressive activity.     

Differential regulation of Jun N-terminal kinase and p38MAP kinase by Galpha12

Based on the findings that the overexpression of the wild-type Galpha(12) (Galpha(12)WT) result in the oncogenic transformation of NIH3T3 cells in a serum-dependent manner, a model system has been established in which the mitogenic and subsequent cell transformation pathways activated by Galpha(12) can be turned on or off by the addition or removal of serum. Using this model system, our previous studies have shown that the stimulation of Galpha(12)WT or the expression of an activated mutant of Galpha(12) (Galpha(12)QL) leads to increased cell proliferation and subsequent oncogenic transformation of NIH3T3 cells, as well as persistent activation of Jun N-terminal kinases (JNKs). In the present studies, we show that the stimulation of Galpha(12)WT or the expression of Galpha(12)QL results in a potent inhibition of p38MAPK, and that the mechanism by which Galpha(12) inhibits p38MAPK activity involves the dual specificity kinases upstream of p38MAPK. The results indicate that Galpha(12) attenuates the activation of MKK3 and MKK4, which are known to stimulate only p38MAPK or p38MAPK and JNK, respectively. The results also suggest that Galpha(12) activates JNKs specifically through the stimulation of the JNK-specific upstream kinase MKK7. These findings demonstrate for the first time that Galpha(12) differentially regulates JNK and p38MAPK by specifically activating MKK7, while inhibiting MKK3 and MKK4 in NIH3T3 cells. Since the stimulation of p38MAPK is often associated with apoptotic responses, our findings suggest that Galpha(12) stimulates cell proliferation and neoplastic transformation of NIH3T3 cells by attenuating p38MAPK-associated apoptotic responses, while activating the mitogenic responses through the stimulation of ERK- and JNK-mediated signaling pathways.