Silibinin synergizes with mitoxantrone to inhibit cell growth and induce apoptosis in human prostate cancer cells

The purpose of these experiments was to assess the synergistic activity of silibinin with chemotherapy agents in clinical use against prostate cancer. Silybin-phytosome, a commercially available formulation containing silibinin, has recently been studied in a phase I clinical trial. The silibinin doses used in the present study are clinically achievable based on the preliminary phase I data. DU145, PC-3 and LNCaP prostate cancer cells were seeded in 96-well plates in triplicate. Twenty-four hours later, silibinin (10, 20 and 40 microM) and either mitoxantrone or docetaxel were added to the designated wells. Seventy-two hours post-treatment, cell viability was determined with a tetrazolium-based assay. The combination index (CI) for determination of a synergistic effect was calculated, with values of <0.9 indicating synergy and values >1.1 antagonism. Apoptosis was also assessed using a luminescent assay after 72 hr of treatment with media alone, silibinin, mitoxantrone, or silibinin plus mitoxantrone. Silibinin showed a synergistic effect with mitoxantrone, as measured by reduction in cell viability. The CI values ranged from 0.413 to 2.650 for the combination of silibinin and mitoxantrone; in contrast, treatment with docetaxel and silibinin showed little or no synergy, with CI values of 0.898-4.469. In concordance with these findings, the addition of silibinin increased the level of apoptosis compared to mitoxantrone alone, particularly in the PC-3 cells. The combination of silibinin and mitoxantrone exhibits a pattern of synergy in reducing cell viability with increased apoptosis. These data are important in the planning of future clinical applications of silibinin.     

Lenalidomide synergizes with dexamethasone to induce growth arrest and apoptosis of mantle cell lymphoma cells in vitro and in vivo

Mantle cell lymphoma (MCL) frequently relapses after therapy and new therapeutic regimens are needed. Lenalidomide (LEN), a thalidomide analogue, displays direct cytotoxicity against MCL cells. This study was undertaken to evaluate the combined therapeutic effect of LEN and dexamethasone (DEX) on MCL. LEN synergized with DEX to induce the growth inhibition and apoptosis of both established MCL cells and freshly isolated MCL cells from refractory or relapsed MCL patients. The synergy was more significant in freshly isolated patients’ MCL cells than established MCL cells. Cell cycle analysis showed that LEN enhanced DEX-induced G₀/G₁ arrest. The effect of the LEN and DEX combination on apoptotic induction was mainly through mitochondrial signaling pathways, as demonstrated by phosphorylation of bcl-2 and up-regulation of proapoptotic proteins Bax, Bad and Bim, and the subsequent activation of caspase-9, caspase-3, and cleavage of PARP. Importantly, the combination of LEN and DEX delayed the tumor growth and improved the survival of MCL-bearing mice. The results support the use of the LEN and DEX combination as a new therapeutic regimen in clinical trials of MCL.     

PRIMA-1(MET) synergizes with cisplatin to induce tumor cell apoptosis

Mutant p53-carrying tumors are often more resistant to chemotherapeutical drugs. We demonstrate here that the mutant p53-reactivating compound PRIMA-1(MET) acts synergistically with several chemotherapeutic drugs to inhibit tumor cell growth. Combined treatment with cisplatin and PRIMA-1(MET) resulted in a synergistic induction of tumor cell apoptosis and inhibition of human tumor xenograft growth in vivo in SCID mice. The induction of mutant p53 levels by chemotherapeutic drugs is likely to increase the sensitivity of tumor cells to PRIMA-1(MET). Thus, the combination of PRIMA-1(MET) with currently used chemotherapeutic drugs may represent a novel and more efficient therapeutic strategy for treatment of mutant p53-carrying tumors.     

Bisphosphonates induce apoptosis in human breast cancer cell lines

Breast cancer has a prodigious capacity to metastasize to bone. In women with advanced breast cancer and bone metastases, bisphosphonates reduce the incidence of hypercalcaemia and skeletal morbidity. Recent clinical findings suggest that some bisphosphonates reduce the tumour burden in bone with a consequent increase in survival, raising the possibility that bisphosphonates may have a direct effect on breast cancer cells. We have investigated the in vitro effects of bisphosphonates zoledronate, pamidronate, clodronate and EB 1053 on growth, viability and induction of apoptosis in three human breast cancer cell lines (MDA-MB-231, Hs 578T and MCF-7). Cell growth was monitored by crystal violet dye assay, and cell viability was quantitated by MTS dye reduction. Induction of apoptosis was determined by identification of morphological features of apoptosis using time-lapse videomicroscopy, identifying morphological changes in nucleis using Hoechst staining, quantitation of DNA fragmentation, level of expression of bcl-2 and bax proteins and identification of the proteolytic cleavage of Poly (ADP)-ribose polymerase (PARP). All four bisphosphonates significantly reduced cell viability in all three cell lines. Zoledronate was the most potent bisphosphonate with IC50 values of 15, 20 and 3 microM respectively in MDA-MB-231, MCF-7 and Hs 578T cells. Corresponding values for pamidronate were 40, 35 and 25 microM, whereas clodronate and EB 1053 were more than two orders of magnitude less potent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleus, time-dependent increase in the percentage of fragmented chromosomal DNA, down-regulation in bcl-2 protein and proteolytic cleavage of PARP, all indicate that bisphosphonates have direct anti-tumour effects on human breast cancer cells.     

Selective cyclooxygenase-2 inhibitors inhibit growth and induce apoptosis of bladder cancer

Selective COX-2 inhibitors such as celecoxib and NS-398 are being evaluated as chemopreventive and therapeutic agents for bladder and other cancers. We investigated the effects of these nonsteroidal anti-inflammatory agents on a panel of bladder cancer cell lines, and assessed their effects on anchorage-dependent and -independent growth, cell cycle, apoptosis and morphology. The human bladder cancer cell lines UM-UC-1, -3, and -6 were assayed for COX-2 expression by Western analysis using a monoclonal antibody to COX-2. UM-UC-1, -3, and -6 cells were grown in the presence of increasing concentrations of NS-398 and celecoxib, and cell growth was quantitated over 7 days by crystal violet elution. The cell lines were treated with NS-398 and celecoxib for 48 h and analyzed by flow cytometry with propidium iodide staining and Br-dUTP staining for apoptosis. Anchorage-independent growth was assessed using an agarose growth assay. Western analysis demonstrated that COX-2 expression in UM-UC-1, -6, and -3 was high, low, and undetectable, respectively. NS-398 and celecoxib produced dose-dependent growth inhibition of UM-UC-1 and -6. Both NS-398 and celecoxib also inhibited anchorage-dependent and -independent growth of UM-UC-3 in a dose-dependent fashion, despite the low basal expression of COX-2 in this cell line. Cell cycle analyses of UM-UC-1 and -6 revealed a 50% reduction in S-phase in the presence of 100 microM NS-398 whereas a smaller reduction in S-phase was noted in UM-UC-3 cells. Furthermore, treatment with 100 microM celecoxib resulted in significant apoptosis in all three cell lines, which was associated with downregulation of Bcl-2. COX-2 selective inhibitors NS-398 and celecoxib produced dose-dependent growth inhibition of bladder cancer cells associated with a significant reduction in S-phase. Induction of apoptosis in all three cell lines by celecoxib was associated with downregulation of Bcl-2. These changes occur independently of COX-2 expression levels suggesting the presence of a COX-2 independent pathway.     

The combination of thymoquinone and paclitaxel shows anti-tumor activity through the interplay with apoptosis network in triple-negative breast cancer

Tumor Biology - Thymoquinone (TQ) is the active ingredient of Nigella sativa which has a therapeutic potential in cancer therapy and prevention. In this study, TQ has been shown to induce specific...     

塞来昔布抑制人类三阴性乳腺癌裸鼠移植瘤生长的实验研究

 【摘要】　目的　探讨塞来昔布（celecoxib）对人类三阴性乳腺癌（TNBC）肿瘤生长及细胞凋亡的影响。方法　32只裸鼠于背部皮下接种人类TNBC细胞株MDA-MB-231,随机分为空白对照组及低、中、高剂量塞来昔布组（25、50、100 mg?kg-1?d-1）。实验结束后,留取移植瘤标本,观察用药前后裸鼠肿瘤体积的变化;流式细胞术（FCM）检测肿瘤细胞凋亡率;免疫组织化学法检测NF-κB p65和p50分子的表达;Western blot法检测凋亡相关分子Caspase-3、Survivin蛋白的表达。结果　塞来昔布治疗组肿瘤体积较对照组均明显减小。中、高剂量塞来昔布治疗组凋亡率分别为（13.58±3.16）％、（21.91±4.75）％,与对照组的（3.15±1.73）％相比差异有统计学意义（t＝6.736,12.151,均P＜0.05）,塞来昔布低、中、高剂量组p65表达阳性率分别为79.3 %、46.7 %、23.9 %,与对照组（89.7 %）相比差异有统计学意义（χ2＝3.312,10.785,15.900,均P＜0.05）。Western blot结果显示,塞来昔布治疗后肿瘤组织中Caspase-3蛋白出现了裂解片段,并且随药物浓度增加,裂解片段表达量逐渐增加。Survivin蛋白随药物浓度增加表达逐渐下调。结论　塞来昔布可以诱导TNBC裸鼠移植瘤细胞凋亡,抑制肿瘤生长,其抗肿瘤作用机制可能部分与抑制p65分子以及下调Survivin蛋白表达有关。     

Gold (III) porphyrin complexes induce apoptosis and cell cycle arrest and inhibit tumor growth in colon cancer

BACKGROUND:

Gold (III) compounds have exhibited favorable antitumor properties both in vitro and in vivo. In a previous study, the authors reported that the novel gold (III) complex 1a (gold 1a) exhibited strong cytotoxicity in some tumor cell lines. In the current study, the effect of gold 1a was investigated on colon cancer cells.

METHODS:

The cytotoxicity of gold 1a was determined by using the 3‐(4,5‐dimethyl‐2‐thihazyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide method. Flow cytometry was used to detect apoptosis and cell cycle. The expression of protein was evaluated by Western blot assay. Tumor growth in vivo was evaluated in nude mice.

RESULTS:

Gold 1a exhibited marked cytotoxic effects in vitro to human colon cancer, and the concentration of drug required to inhibit cell growth by 50% compared with control (IC₅₀) values ranged from 0.2 μM to 3.4 μM, which represented 8.7‐fold to 20.8‐fold greater potency than that of cisplatin. Gold 1a significantly induced apoptosis and cell cycle arrest and cleaved caspase 3, caspase 7, and poly(ADP‐ribose) polymerase; released cytochrome C, and up‐regulated p53, p21, p27, and Bax. In vivo, intraperitoneal injection of gold 1a at doses of 1.5 mg/kg and 3.0 mg/kg significantly inhibited tumor cell proliferation, induced apoptosis, and suppressed colon cancer tumor growth. An acute toxicology study indicated that gold 1a at effective antitumor concentrations did not cause any toxic side effects in mice.

CONCLUSIONS:

The current results suggested that gold 1a may be a new potential therapeutic drug for colon cancer. Cancer 2009. © 2009 American Cancer Society.     

转染beclin 1基因诱导自噬对三阴性乳腺癌BT-549细胞生长的影响

目的探讨转染beclin1基因诱导三阴性乳腺癌BT-549细胞发生自噬及其对细胞生长的影响。方法将BT-549细胞分为4组,包括空白组、空载体组、脂质体组和目的基因组,并以MDA—MB-231细胞作为阳性对照组。采用Lipofectamine^TM 2000携带含有beclin1基因的质粒转染BT-549细胞,分别于转染后24、36、48、60h用荧光显微镜观察转染效率;于转染后48h利用RT-PCR检测各组细胞beclin 1 mRNA表达情况;转染后各组细胞分别在含10％胎牛血清1640培养基和无胎牛血清1640培养基内培养,并分别于24、48、72、96h和12、24、36、48h采用MTT法检测细胞的增殖率和存活率;各组细胞在无胎牛血清1640培养基内培养24h后,采用流式细胞仪检测其细胞凋亡和细胞周期情况;各组细胞在无胎牛血清1640培养基内生长36h后,经吖啶橙染色观察并比较发生自噬的细胞数。MTT数据按重复测量的方差分析进行统计,其余计量资料按单因素方差分析进行统计,计数资料按照R×C表资料的χ^2检验进行统计分析。结果48h时携带目的基因的质粒经脂质体转染BT-549细胞的效率最高;目的基因组细胞beclin 1 mRNR水平显著高于空白组、脂质体组、空载体组和阳性对照组（P〈0．01）;在含10％胎牛血清1640培养基内,目的基因组细胞的增殖率显著低于空白组（F=112．1,P=0．00）,而在无胎牛血清1640培养基内,目的基因组细胞36h和48h的存活率显著高于空白组（F=37．5,P=0．00）;目的基因组细胞的凋亡比例增加（F=3914．4,P=0．00）,且细胞处于G0／G1期的比例升高（F=258．8,P=0．00）;目的基因组细胞发生自噬的数量显著增加（χ^2=7．7,P=0．00）。结论向三阴性乳腺癌BT-549细胞转染beclin 1基因,可提高细胞的自噬水平。该作用可抑制细胞在正常培养环境下的增殖,但对低营养环境下的细胞存活具有保护作用。     

硼替佐米与吡柔比星协同抑制T细胞淋巴瘤Hut-78细胞增殖的体外研究

 目的　探讨硼替佐米（BTZ）对人类皮肤T细胞淋巴瘤Hut-78细胞系的细胞毒作用及其与吡柔比星（THP）联合应用的抗肿瘤效果。方法　采用MTT法观察BTZ、THP单药及两药联合不同给药顺序对Hut-78细胞增殖的抑制作用,利用中效方程分析两药是否存在协同效应。结果　BTZ、THP单药均能有效抑制Hut-78细胞的生长,且呈时间和剂量依赖性,培养48 h的半数抑制浓度分别为8.53 nmol／L和2515.27 μg／L。两药联合具有显著的抗肿瘤活性,当增殖抑制率＞50 %时,THP序贯BTZ呈现拮抗效应,而BTZ与THP同时及BTZ序贯THP均具有协同效应,后者的抗肿瘤作用更为显著。结论　BTZ单药及其与THP联合体外可有效抑制T细胞淋巴瘤细胞增殖。BTZ是一种很有前景的治疗T细胞淋巴瘤的靶向药物,为BTZ与THP联合治疗T细胞淋巴瘤的临床研究提供了实验依据。     

TRAIL induces apoptosis in triple-negative breast cancer cells with a mesenchymal phenotype

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in some but not all breast cancer cell lines. Breast cancers can be divided into those which express the estrogen (ER) and progesterone (PR) receptors, those with HER-2 amplification, and those without expression of ER, PR, or HER-2 amplification (referred to as basal or triple-negative breast cancer). We tested a panel of 20 breast cancer cell lines representing the different types of breast cancer to evaluate if the molecular phenotype of the breast cancer cells determined their response to TRAIL. The most striking finding was that eight of eleven triple-negative cell lines are sensitive to TRAIL-mediated apoptosis. The eight TRAIL-sensitive triple-negative cell lines have a mesenchymal phenotype while the three TRAIL-resistant triple-negative cell lines have an epithelial phenotype. Two of five cell lines with HER-2 amplification were sensitive to TRAIL and none of the five ER positive cell lines were sensitive. RNAi-mediated knockdown of TRAIL receptor expression demonstrated that TRAIL Receptor 2 (TRAIL-R2) mediates the effects of TRAIL, even when both TRAIL-R1 and TRAIL-R2 are expressed. Finally, inhibition of EGFR, expressed in both TRAIL-sensitive and TRAIL-resistant triple-negative breast cancer cell lines, using a small molecule tyrosine kinase inhibitor (AG1478), enhanced TRAIL-induced apoptosis in TRAIL-sensitive cell lines but did not convert resistant cells into TRAIL-sensitive cells. Together, these findings suggest that a subset of triple-negative breast cancer, those with mesenchymal features, may be the most likely to benefit from TRAIL targeted therapy. These findings could form the basis to select breast cancer patients for clinical trials of TRAIL-R2 ligands. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. S. R. Davis and J. G. Pumphrey contributed equally to this work.     

hTERT启动子介导肿瘤凋亡研究进展

在细胞永生化和肿瘤发展过程中,端粒酶的激活是一个关键性的步骤.人类端粒酶逆转录酶(hTERT)基因的表达是端粒酶激活的限速步骤.现综述hTERT基因启动子介导肿瘤凋亡的最新研究进展.