Induction of apoptosis and inhibition of c-erbB-2 in breast cancer cells by flavopiridol.

Flavopiridol is a flavone that inhibits several cyclin-dependent kinases and exhibits potent growth-inhibitory activity against a number of human tumor cell lines, both in vitro and when grown as xenografts in mice. It is presently being investigated as a novel antineoplastic agent in the primary screen conducted by the Developmental Therapeutics Program, National Cancer Institute. Because breast cancer is the most common cancer and second leading cause of cancer-related deaths in women in the United States, we investigated whether flavopiridol could be an effective agent against a series of isogenic breast- cancer cell lines having different levels of erbB-2 expression and differential invasion and metastatic characteristics. Flavopiridol was found to inhibit the growth of MDA-MB-435 (parental) and 435.eB (stable transfectants) cells that were established by transfecting c-erbB-2 cDNA into MDA-MB-435. Induction of apoptosis was also observed in these cell lines when treated with flavopiridol, as measured by DNA laddering, PARP, and CPP32 cleavages. We also found modest up-regulation of Bax and down-regulation of Bcl-2, but there was a significant down-regulation of c-erbB-2 in flavopiridol-treated cells. Gelatin zymography showed that flavopiridol inhibits the secretion of matrix metalloproteinase (MMP; MMPs 2 and 9) in the breast cancer cells and that the inhibition of c-erbB-2 and MMPs may be responsible for the inhibition of cell invasion observed in flavopiridol-treated cells. Collectively, these molecular effects of flavopiridol, however, were found to be independent of c-erbB-2 overexpression, suggesting that flavopiridol may be effective in all breast cancer. From these results, we conclude that flavopiridol inhibits the growth of MDA-MB-435 breast cancer cells, induces apoptosis, regulates the expression of genes, and inhibits invasion and, thus, may inhibit metastasis of breast cancer cells. These findings suggest that flavopiridol may be an effective chemotherapeutic or preventive agent against breast cancer.     

Induction of apoptosis by thiosulfinates in primary human prostate cancer cells

Thiosulfinates, a substance of Allium tuberosum L., is a known folk medicine that has been extensively used in diet to treat diseases. In the present study, we have evaluated the effect of thiosulfinates from Allium tumberosum L. on proliferation of metastasis (DU145) and primary malignant tumor (RC-58T/h/SA#4)-derived human prostate cancer cells. Thiosulfinates decrease viable cell numbers in a dose- and time-dependent manner and induce apoptosis. The apoptosis induced by thiosulfinates is associated with the activation of initiator caspase-8, and -9, and the effector caspase-3. Thiosulfinates stimulated Bid cleavage, indicating that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. Thiosulfinates decreased the expression of the anti-apoptotic protein Bcl-2, and increased the expression of the pro-apoptotic protein Bax. Thiosulfinates also increased the expression of AIF, a caspase-independent mitochondrial apoptosis factor, in RC-58T/h/#4 cells and induced DNA fragmentation and chromatin condensation. These results indicate that thiosulfinates from Allium tuberosum L. inhibit cell proliferation by inducing apoptosis in RC-58T/h/#4 cells which may be mediated via both caspase-dependent and caspase-independent pathways.     

Growth inhibition and apoptosis of myeloma cells by the CDK inhibitor flavopiridol.

Although myeloma shows responsiveness in intensive chemotherapy, overall survival remains less than 40% at 2 years. Since myeloma appears to be dependent on cytokines, such as IL-6, we hypothesized that targeting signal transduction molecules could effectively treat myeloma. Two myeloma cell lines U266 and RPMI-8226 and CD38+ myeloma cells were studied by immune complex kinase assay or anti-phosphotyrosine blot for evidence of constitutive activation of tyrosine kinases. Growth arrest and apoptosis were evaluated in these two cell lines following their treatment with specific kinase inhibitors. We found that a variety of Src and Janus kinases were present and constitutively active in U266 and RPMI-8226 cells. Inhibitors of both Src and Janus kinases were inferior to the cyclin-dependent kinase inhibitor, flavopiridol, in inducing both growth arrest with GI50 of 100 nM and apoptosis in both cell lines and CD38+ myeloma cells. Although, flavopiridol did not affect cyclin D1 and cyclin A levels, it inhibited Mcl-1 and Bcl-2 protein levels and cyclin-dependent kinase 2 activity. Flavopiridol is a well-tolerated drug, currently in phase I-II trials for a variety of tumors. A clinical trial using flavopiridol should be performed in patients with myeloma. Its mechanism of action may involve targets other than the cyclin-dependent kinases.     

Induction of androgen receptor-dependent apoptosis in prostate cancer cells by the retinoblastoma protein

Re-expression of a tumor suppressor in tumor cells that lack it is an effective way to study its functional activities. However, because tumor cells contain multiple mutations, tumor suppressor functions that are dependent on (an)other regulators are unlikely to be identified by its re-expression alone if the other regulators are also mutated. In this study, we show that re-expression of retinoblastoma (RB) together with the androgen receptor (AR) in RB- and AR-deficient prostate cancer DU-145 cells resulted in an apoptotic activity, acting through the mitochondria damage-initiated caspase activation pathway, which was not present when RB, or the AR, was re-expressed alone. The ability of RB + AR to induce mitochondria damage was dependent on the proapoptotic proteins Bax and Bak and could be blocked by the antiapoptotic protein Bcl-x(L). Coexpressed AR did not detectably change RB's regulation of E2F and cell cycle progression in culture. On the other hand, coexpressed RB could activate the transactivation activity of the AR in an androgen-depleted media. Although androgen induced greater AR transactivation activity in this condition, it did not induce apoptosis in the absence of coexpressed RB. Analysis of mutants of RB and the AR indicated that intact pocket function of RB and the transactivation activity of the AR were required for RB + AR-induced apoptosis. These results provide direct functional data for an AR-dependent apoptosis-inducing activity of RB and highlight the importance of cell type-specific regulators in obtaining a more complete understanding of RB.     

Induction of autophagy-dependent apoptosis by the survivin suppressant YM155 in prostate cancer cells

In this study, we demonstrated that YM155, a novel survivin suppressant, induced both apoptosis, and autophagy that was shown by conversion of cytosolic-associated protein light chain 3 (LC3I) into autophagosome-associated form (LC3II) and a punctate fluorescence pattern of an ectopic GFP-LC3 protein. The lysosomal inhibitor chloroquine further accumulated YM155-induced LC3II, indicating an increase of autophagic flux. Ectopic expression of survivin significantly attenuated YM155-induced apoptosis and autophagy, whereas survivin siRNA induced autophagy. Furthermore, inhibition of either early or late events of autophagy attenuated YM155-induced apoptosis, demonstrating that induction of autophagy proceeds apoptosis. In conclusion, suppression of survivin by YM155 induces autophagy-dependent apoptosis, and YM155-induced autophagy plays a pro-apoptotic role thereby unveiling a novel mechanism of YM155 in prostate cancer cells.     

RNA interference-mediated silencing of the acetyl-CoA-carboxylase-alpha gene induces growth inhibition and apoptosis of prostate cancer cells

Overexpression of lipogenic enzymes is a common characteristic of many cancers. Thus far, studies aimed at the exploration of lipogenic enzymes as targets for cancer intervention have focused on fatty acid synthase (FAS), the enzyme catalyzing the terminal steps in fatty acid synthesis. Chemical inhibition or RNA interference (RNAi)-mediated knockdown of FAS consistently inhibits the growth and induces death of cancer cells. Accumulation of the FAS substrate malonyl-CoA has been implicated in the mechanism of cytotoxicity of FAS inhibition. Here, using RNAi technology, we have knocked down the expression of acetyl-CoA carboxylase-alpha (ACC-alpha), the enzyme providing the malonyl-CoA substrate. Silencing of the ACC-alpha gene resulted in a similar inhibition of cell proliferation and induction of caspase-mediated apoptosis of highly lipogenic LNCaP prostate cancer cells as observed after FAS RNAi. In nonmalignant cells with low lipogenic activity, no cytotoxic effects of knockdown of ACC-alpha or FAS were observed. These findings indicate that accumulation of malonyl-CoA is not a prerequisite for cytotoxicity induced by inhibition of tumor-associated lipogenesis and suggest that in addition to FAS, ACC-alpha is a potential target for cancer intervention.     

Adenovirus-mediated inhibition of NF-kappaB confers chemo-sensitization and apoptosis in prostate cancer cells

Acquirement of multi-drug resistance by tumor cells represents a major obstacle in the management of prostate cancer. Such resistance was demonstrated in the androgen-independent DU-145 cells in response to paclitaxel and the mechanisms by which these cell develops resistance was not understood. The objective of this study was to examine whether abrogation of the constitutively active NF-kappaB in the chemoresistant, androgen independent DU-145 prostate cancer cells will enhance their sensitivity to cytototoxic agents. Inhibition of NF-kappaB by a dominant negative super-repressor IkappaB mutant adenoviral construct enhanced the apoptotic potentials of paclitaxel and rhTNF-alpha in these cells. Using reporter assays and RT-PCR analysis, we demonstrate that paclitaxel-induced cell death was associated with an increase in NF-kappaB activation and MDR-1 gene expression. Abrogation of these effects by the dominant negative IkappaB adenoviral construct suggests that induction and/or constitutive activation of NF-kappaB can block the paclitaxel-induced apoptotic signaling pathways in this cell line, possibly by increasing the expression of anti-apoptotic and MDR-1 gene products, leading to development of chemoresistance in these cells. We conclude that inhibition of NF-kappaB activation may have therapeutic implications for prostate cancer.     

Induction of apoptosis of integrin-expressing human prostate cancer cells by cyclic Arg-Gly-Asp peptides.

Prostate cancer is the second most common cause of cancer deaths among men in the United States. We have investigated the effect of cyclo-(Arg-Gly-Asp-D-Phe-Val; cRGDfV), Arg-Gly-Asp, or Arg-Gly-Asp-Ser, on survival of human prostate cancer (LNCaP and PC-3) and normal (HEL) cells in vitro. Addition of cRGDfV (20 microg/ml) but not the linear Arg-Gly-Asp or Arg-Gly-Asp-Ser peptide induced significant (approximately 84%) killing of LNCaP cells expressing alphavbeta3 integrins on their surfaces. In contrast, none of these peptides had any major effect on the growth of PC-3 or HEL cells, which express little alphavbeta3 integrin on their surfaces. Treatment of LNCaP but not of PC-3 or HEL cells with cRGDfV resulted in cleavage of focal adhesion kinase, a key player in integrin-mediated signal transduction pathway. The evidence we present here suggests that the killing of LNCaP cells after cRGDfV treatment was attributable to apoptosis or programmed cell death. This is evidenced by activation of at least two caspases (caspase-3 and caspase-9) as detected by cleavage of poly(ADP-ribose) polymerase and partial blocking of apoptosis by a selective inhibitor of caspase-9. Our results suggest that cRGDfV may be an effective treatment for some human prostate cancers by inducing apoptosis through interference with the regulation of integrin/focal adhesion kinase-mediated signal transduction pathway necessary for cell survival.     

Induction of apoptosis and inhibition of human gastric cancer MGC-803 cell growth by arsenic trioxide

Arsenic trioxide (As2O3), used to treat human diseases for centuries in traditional Chinese medicine, has been identified as a very effective antileukaemic agent, but its effect on solid tumours which could be more suitable for clinical treatment with arsenic compounds is still unknown. In this study, we investigated the in vitro effect of As2O3 at concentrations of 0.01-1 microM against six human malignant cell lines, MGC-803, HIC, MCF-7, HeLa, BEL-7402 and A549 cells. As2O3 inhibited growth and induced apoptosis in these malignant cells at varying degrees, in a time dose-dependent manner. The most marked effects were seen in the gastric cancer cell line, MGC-803. In contrast, minimal growth inhibition and induction of apoptosis occurred in human embryonic pulmonary cells following treatment with As2O3 found at the same concentrations. Changes in intracellular Ca2+, following As2O3 treatment were measured by Ca2+ sensitive fluorescent probe Indo-1/AM in flow cytometric assays. The increase in intracellular Ca2+ correlated with the sensitivity of these cells to As2O3, possibly indicating that a critical intracellular Ca2+ signal transduction pathway could be involved in As2O3-mediated cell-death and its selectivity. The marked sensitivity of MGC-803 cells in vitro suggests that As2O3 may be a potential antigastric cancer agent.     

Docetaxel-induced growth inhibition and apoptosis in androgen independent prostate cancer cells are enhanced by 1alpha,25-dihydroxyvitamin D3

Pre-treatment with high-dose 1alpha,25-dihydroxyvitamin D(3) (1,25-VD) enhanced the antitumor activity of docetaxel in the androgen-independent prostate cancer cell line, PC-3. The effect manifested as an increasing population of apoptotic cells and amount of pro-apoptotic protein, Bax, under combined treatment compared with single treatment of either 1,25-VD or docetaxel alone. We further demonstrated that pre-treatment with 1,25-VD reduced the expression of multidrug resistance-associated protein-1 at both the mRNA and protein levels. This suggests pre-treatment with 1,25-VD can potentiate cytotoxicity of docetaxel in PC-3 due to 1,25-VD reducing multidrug resistance-associated protein-1 expression.     

Zoledronic acid mediates Ras-independent growth inhibition of prostate cancer cells

Zoledronic acid (ZOL), the most potent known bisphosphonate, is clinically efficacious against advanced prostate cancer, although the molecular mechanism by which bisphosphonates prevent prostate cancer cell growth remains unknown. Because Ras is the most thoroughly characterized member of the small G-proteins involved in the regulation of many cellular functions including several oncogenic pathways, the aim of this study was to clarify whether Ras is the molecular target of ZOL in prostate cancer cells. The prostate cancer cell lines PC-3, DU145, and LNCaP were used. Cell proliferation was determined by a modified MTT assay. Geranylgeranyol (GGOH) and famesol (FOH) were used as analogues of geranylgeranyl-pyrophosphate and farnesyl-pyrophosphate, respectively. Changes in expression and/or membrane localization of Ras, Rap1, and phosphorylated MAPK were evaluated by Western blotting. ZOL mediated growth inhibition of prostate cancer cells in a dose- and time-dependent manner. The ZOL-induced growth inhibitory effect was circumvented by the addition of GGOH. In contrast, FOH did not reverse the growth inhibitory effect of ZOL. The amount of membrane-anchored Ras was clearly independent of ZOL-mediated growth inhibition. Unexpectedly, ZOL induced N- and H-Ras expression of the cytosolic fraction. Ras does not appear to be the molecular target for ZOL-induced growth inhibition. Prevention of geranylgeranylation rather than farnesylation is an important therapeutic target in prostate cancer.     

Mechanisms of nordihydroguaiaretic acid-induced growth inhibition and apoptosis in human cancer cells

Lipoxygenase metabolites of arachidonic acid can act as growth promoting factors for various cancer cell lines. Here we demonstrate that the 5-lipoxygenase inhibitor nordihydroguaiaretic acid potently inhibits anchorage-independent growth of human pancreatic and cervical cancer cells in soft agar and delays growth of pancreatic and cervical tumours established in athymic mice. Furthermore, nordihydroguaiaretic acid induces apoptosis of these cancer cells in vitro and in vivo. Potential mechanisms mediating these effects of nordihydroguaiaretic acid were examined. Nordihydroguaiaretic acid had no inhibitory effect on growth and survival signals such as tyrosine phosphorylation of the epidermal growth factor receptor or basal and growth factor-stimulated activities of extracellular signal-regulated kinase 1/2, p70(s6k) and AKT but selectively inhibited expression of cyclin D1 in the cancer cells. In addition, treatment with nordihydroguaiaretic acid lead to a disruption of the filamentous actin cytoskeleton in human pancreatic and cervical cancer cells which was accompanied by the activation of Jun-NH(2)-terminal kinase and p38(mapk). Similar effects were obtained by treatment of the cancer cells with cytochalasin D. These results suggest that nordihydroguaiaretic acid induces anoikis-like apoptosis as a result of disruption of the actin cytoskeleton in association with the activation of stress activated protein kinases. In conclusion, nordihydroguaiaretic acid could constitute a lead compound in the development of novel therapeutic agents for various types of cancer.     

Sphingosine kinase-1 inhibition sensitizes curcumin-induced growth inhibition and apoptosis in ovarian cancer cells

Recent published studies suggest that increasing levels of ceramides enhance the chemo-sensitivity of curcumin. Using in vitro approaches, we analyzed the impact of sphingosine kinase-1 (SphK-1) inhibition on ceramide production, and evaluated SphK1 inhibitor II (SKI-II) as a potential curcumin chemo-sensitizer in ovarian cancer cells. We found that SphK1 is overexpressed in ovarian cancer patients' tumor tissues and in cultured ovarian cancer cell lines. Inhibition of SphK1 by SKI-II or by RNA interference (RNAi) knockdown dramatically enhanced curcumin-induced apoptosis and growth inhibition in ovarian cancer cells. SKI-II facilitated curcumin-induced ceramide production, p38 activation and Akt inhibition. Inhibition of p38 by the pharmacological inhibitor (SB 203580), a dominant-negative expression vector, or by RNAi diminished curcumin and SKI-II co-administration-induced ovarian cancer cell apoptosis. In addition, restoring Akt activation introducing a constitutively active Akt, or inhibiting ceramide production by fumonisin B1 also inhibited the curcumin plus SKI-II co-administration-induced in vitro anti-ovarian cancer effect, suggesting that ceramide accumulation, p38 activation and Akt inhibition are downstream effectors. Our findings suggest that low, well-tolerated doses of SKI-II may offer significant improvement to the clinical curcumin treatment of ovarian cancer.     

Sodium butyrate and tributyrin induce in vivo growth inhibition and apoptosis in human prostate cancer

Histone deacetylase inhibitors (HDACs) are known to exhibit antiproliferative effects on various carcinoma cells. In this study, the in vivo efficiency of two HDACs, sodium butyrate and tributyrin, on prostate cancer growth inhibition were investigated. To gain an insight into the possible underlying pathways, cell culture experiments were performed focusing on the expression of p21, Rb and c-myc. For in vivo testing, prostate cancer cell lines (PC3 and TSU-Pr1) were seeded on the chorioallantois membrane (CAM) and implanted in a xenograft model using nude mice. Standard Western blot analysis was performed for protein expression of p21, Rb and c-myc in HDAC-treated vs untreated prostate cancer cells. Both sodium butyrate and tributyrin had a considerable treatment effect on microtumours on the chicken egg at already very low concentrations of 0.1 mM. Tributyrin-treated tumours showed the strongest effect with 38% apoptotic nuclei in the prostate cancer cell line PC3. In the mouse model, there was almost no difference between sodium butyrate and tributyrin. In untreated animals the tumours were almost double the size 4 weeks after implantation. Tumours of the treatment groups had a significantly lower percentage of Ki-67-positive-stained nuclei. As demonstrated by Western blot analysis, these effects seem to be independent of p53 status and a pathway via p21-Rb-c-myc is possibly involved. In this study we have demonstrated a substantial in vivo treatment effect, which can be induced by the application of sodium butyrate or the orally applicable tributyrin in human prostate cancer. The given results may provide the rationale to apply these drugs in well-controlled clinical trials in patients being at high risk of recurrence after specific therapy or in patients with locally or distant advanced prostate cancer.     

Statin induces apoptosis and cell growth arrest in prostate cancer cells.

Statins are a class of low molecular weight drugs that inhibit the rate-limiting enzyme of the mevalonate pathway 3-hydroxy-3-methylglutaryl-CoA reductase. Statins have been approved and effectively used to control hypercholesterolemia in clinical setting. Recent study showed statin's antitumor activity and suggested a potential role for prevention of human cancers. In this study, we did cell viability, DNA fragmentation, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays to evaluate the action of statins on prostate cancer cells and used Western blotting and RhoA activation assay to investigate the underlying molecular mechanism of action. Our data showed that lovastatin and simvastatin effectively decreased cell viability in three prostate cancer cell lines (PC3, DU145, and LnCap) by inducing apoptosis and cell growth arrest at G(1) phase. Both lovastatin and simvastatin induced activation of caspase-8, caspase-3, and, to a lesser extent, caspase-9. Both statins suppressed expression of Rb, phosphorylated Rb, cyclin D1, cyclin D3, CDK4, and CDK6, but induced p21 and p27 expression in prostate cancer cells. Furthermore, lovastatin and simvastatin suppressed RhoA activation and c-JUN expression, but not cyclooxygenase-2 expression. Our data showed that the antitumor activity of statins is due to induction of apoptosis and cell growth arrest. The underlying molecular mechanism of statin's action is mediated through inactivation of RhoA, which in turn induces caspase enzymatic activity and/or G(1) cell cycle. Future studies should focus on examining statins and other apoptosis-inducing drugs (e.g., cyclooxygenase-2 inhibitors or curcumin) together to assess their efficacy in prevention of prostate cancer.     

Induced apoptosis in human prostate cancer cells by blocking of vascular endothelial growth factor by siRNA

Introduction

Vascular endothelial growth factor (VEGF) regulates several cell functions including; proliferation, differentiation, permeability, vascular tone, and the production of vasoactive molecules. The purpose of this study was to evaluate the potency of specific short-interfering RNA (siRNA) to suppress human VEGF expression by siRNA and investigate the effects of VEGF down-regulation on the cell proliferation and apoptosis of the human prostate cancer cell lines DU-145.

Methods

Transfection was performed using X-tremeGENE siRNA transfection reagent. At different time intervals, transfected cells were harvested and total RNA was extracted for RT-PCR. The VEGF content in supernatants were measured by ELISA. Inhibition of cell growth by hVEGF-siRNA was measured by using cell proliferation ELISA BrdU assay. Apoptotic cells were evaluated by using annexin-V-FITC apoptotic detection method.

Results

Transfection of hVEGF-siRNA resulted in statistically significant inhibition of hVEGF-mRNA that in turn caused a marked reduction in the expression of hVEGF. The cell growth was assessed every 24?h for 4?days after siRNA treatment resulted in a marked inhibition of cell proliferation as compared to scramble siRNA. The results of apoptosis showed that approximately 15?% of the cells treated with control-siRNA manifested evident apoptotic changes after 24?hpt, whereas DU-145 cells treated with hVEGF-siRNA significantly were positive, that is to say, 53?% at 72?hpt 23.9?±?2.78?% (P?<?0.001) and 13?±?1.57?% at 96?hpt.

Conclusion

Our findings indicate that siRNA are effective in eliciting the RNAi pathway in cancerous cells and that specific siRNA efficiently down-regulate VEGF expression. They could decrease VEGF production and induce apoptosis, which may also be linked to the inhibition of cancerous cell proliferation. Therefore, it can be concluded that siRNA-mediated suppression of VEGF represents a powerful tool against prostate cancer cell proliferation. VEGF down-regulation exerts a direct anti-apoptotic function in the DU-145 cell lines and promises the development of drugs for cancer therapy.     

Induction of apoptosis in human lung cancer cells by curcumin

Curcumin, a phenolic compound from the rhizome of the plant Curcuma longa has anti-inflammatory, antioxidant and anti-cancer activities. Although the precise mode of action of this compound is not yet elucidated, studies have shown that chemo-preventive action of curcumin might be due to its ability to induce apoptosis and to arrest cell cycle. This study investigated the cellular and molecular changes induced by curcumin leading to the induction of apoptosis in human lung cancer cell lines-A549 and H1299. A549 is p53 proficient and H1299 is p53 null mutant. The lung cancer cells were treated with curcumin (0-160 microM) for 12-72 h. Curcumin inhibited the growth of both the cell lines in a concentration dependent manner. Growth inhibition of H1299 cell lines was both time and concentration dependent. Curcumin induced apoptosis in both the lung cancer cell lines. A decrease in expression of p53, bcl-2, and bcl-X(L) was observed after 12 h exposure of 40 microM curcumin. Bak and Caspase genes remained unchanged up to 60 microM curcumin but showed decrease in expression levels at 80-160 microM. The data also suggest a p53 independent induction of apoptosis in lung cancer cells.     

Induction of apoptosis by flavopiridol in human neuroblastoma cells is enhanced under hypoxia and associated with N-myc proto-oncogene down-regulation.

PURPOSE: Neuroblastoma is the most common extracranial solid tumor of children that arises from the sympathetic nervous system. Survival rates for neuroblastoma patients is low despite intensive therapeutic intervention, and the identification of new effective drugs remains a primary goal. The cyclin-dependent kinase inhibitor, flavopiridol, has demonstrated growth-inhibitory and cytotoxic activity against various tumor types. Our aim was to investigate flavopiridol effects on advanced-stage, N-myc proto-oncogene (MYCN)-amplified human neuroblastomas and the modulation of its activity by hypoxia, a critical determinant of tumor progression and a major challenge of therapy. EXPERIMENTAL DESIGN: Cell viability was monitored by 3-(4,5 dimethyl-2 thiazolyl)-2,5 diphenyl-2H tetrazolium bromide (MTT) and trypan blue dye exclusion assays; DNA synthesis was assessed with the bromodeoxyuridine pulse-labeling technique; apoptosis was studied by Giemsa staining, DNA fragmentation, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling reaction, flow cytometric determination of hypodiploid DNA content, and evaluation of caspase activity and cytochrome c (CytC) release; MYCN expression was determined by Northern and Western blotting. RESULTS: Flavopiridol caused dose- and time-dependent decreases in neuroblastoma viability by inducing apoptosis, as confirmed by morphologic and biochemical criteria. Cell death was preceded by DNA synthesis inhibition and G1-G2 arrest, reversed by the pancaspase inhibitor, zVAD-fmk, and associated with caspase-3 and -2 activation and CytC increase. Moreover, flavopiridol strongly down-regulated MYCN mRNA and protein expression. Exposure to hypoxia enhanced both the extent of apoptosis and flavopiridol effects on CytC, caspase 3, and MYCN. CONCLUSIONS: These results indicate that flavopiridol has growth-inhibitory and apoptotic activity against advanced-stage neuroblastomas in vitro and is worthy of further investigation for the treatment of this disease.     

全反式视黄酸对癌细胞凋亡的诱导

探讨全反式视黄酸对乳腺癌细胞生长抑制对细胞凋亡诱导的机理。方法；采用荧光显微术，流式细胞，ＭＴＴ测定和ＲＮＡ印迹等方法，检测细胞凋亡，细胞生长和ｍＲＮＡ的表达水平。结果；１）ＡＴＲＡ能够有效地诱导ＺＲ－７５－１乳腺癌细胞凋亡，但不能诱导ＭＤＡ－ＭＢ－２３１乳腺癌细胞凋亡。２）ＡＴＲＡ能够有效地抑制ＺＲ－７５－１细胞的生长，但不能抑制ＭＤＡ－ＭＢ－２３１细胞生长 ；３）ＡＴＲＡ能够诱导ＺＲ－７５－１