Cotylenin A,a differentiation-inducing agent,and IFN-alpha cooperatively induce apoptosis and have an antitumor effect on human non-small cell lung carcinoma cells in nude mice

Cotylenin A, a novel inducer of the differentiation of leukemia cells, and IFN-alpha synergistically inhibited the growth of and induced apoptosis in several human non-small cell lung carcinoma cell lines. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor DR5 were the early genes induced by the combination of cotylenin A and IFN alpha in lung carcinoma cells. Neutralizing antibody to TRAIL inhibited apoptosis, suggesting that cotylenin A and IFN alpha cooperatively induced apoptosis through the TRAIL signaling system. This combined treatment preferentially induced apoptosis in human lung cancer cells while sparing normal lung epithelial cells and significantly inhibited the growth of human lung cancer cells as xenografts without apparent adverse effects, suggesting that this combination may have therapeutic value in treating lung cancer.     

Cotylenin A, a new differentiation inducer, and rapamycin cooperatively inhibit growth of cancer cells through induction of cyclin G2

Cotylenin A, a plant growth regulator, and rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), are potent inducers of differentiation of myeloid leukemia cells. Recently, we found that cotylenin A and rapamycin effectively inhibited the proliferation of several human breast cancer cell lines including MCF-7. Herein, we demonstrate that cotylenin A and rapamycin rapidly and markedly induced the cyclin G2 gene expression in several cancer cells including MCF-7 cells. The growth arrest of the MCF-7 cells at the G1 phase, induced by the treatment with cotylenin A and rapamycin or the culture in low serum medium, markedly induced the cyclin G2 gene expression. Anticancer drugs including doxorubicin, etoposide and 5-fluorouracil also induced cyclin G2 expression during induction of growth arrest of the MCF-7 cell at the G1 phase or G2/M phase. Ectopically inducible cyclin G2 expression potently inhibited the proliferation of MCF-7 cells. Furthermore, cyclin G2 knockdown induced by cyclin G2 small interfering RNA markedly reduced the potency of cotylenin A plus rapamycin to induce growth inhibition. Taken together, our results suggest that cotylenin A and rapamycin induce inhibition of cancer cell growth through the induction of cyclin G2. ( Cancer Sci 2008; 99: 1693–1698)     

Cotylenin A-induced differentiation is independent of the transforming growth factor-beta signaling system in human myeloid leukemia HL-60 cells

Cotylenin A, which has been isolated as a plant growth regulator, potently induces the differentiation of human myeloid leukemia cells. Treatment of HL-60 cells with a combination of transforming growth factor (TGF)-beta and 1alpha, 25-dihydroxyvitamin D(3) (VD3) resulted in increased differentiation compared to separate treatments, but TGF-beta did not affect the cotylenin A-induced differentiation of HL-60 cells. It is possible that the signal transduction pathway used by cotylenin A for inducing the differentiation of leukemia cells is the same as that used by TGF-beta. However, cotylenin A did not affect the expression of TGF superfamily or Smad genes in HL-60 cells. Treatment with neutralizing anti-TGF-beta antibody or an inhibitor of TGF-beta signaling did not inhibit cotylenin A-induced differentiation, although VD3-induced differentiation was significantly suppressed by these treatments. The subcellular distribution of Smad3 was also unaffected by cotylenin A. These results suggest that the cotylenin A-induced differentiation of leukemia cells is independent of the TGF-beta signaling system, although TGF-beta acts as an autocrine mediator of the growth arrest and differentiation of leukemia cells induced by VD3 and other inducers.     

Induction of differentiation of myeloid leukemia cells in primary culture in response to lithocholic acid acetate, a bile acid derivative, and cooperative effects with another differentiation inducer, cotylenin A

Lithocholic acid (LCA) acetate induced the differentiation of human leukemia cells. Treatment with a combination of LCA acetate and cotylenin A, an inducer of the differentiation of leukemia cells, was more effective than that with LCA acetate or cotylenin A alone at inducing monocytic differentiation. LCA acetate activated mitogen-activated protein kinase (MAPK) before inducing differentiation. Cotylenin A did not activate MAPK, suggesting that cotylenin A has a different mode of action. The cooperative effects of LCA acetate and cotylenin A on inducing differentiation were, at least partly, due to the enhancement of LCA acetate-induced MAPK activation by cotylenin A.     

Treatment of human promyelocytic leukemia in the SCID mouse model with cotylenin A,an inducer of myelomonocytic differentiation of leukemia cells

Cotylenin A has differentiation-inducing activity in human myeloid leukemia cell lines and leukemic cells that were freshly isolated from acute myeloid leukemia (AML) patients in primary culture. Injection of the human promyelocytic leukemia cell line NB4 into SCID mice resulted in the death of all mice due to leukemia. Administration of cotylenin A significantly prolonged the survival of mice inoculated with NB4 cells. In an in vivo analysis, cotylenin A induced the differentiation of leukemia cells in a retinoid-resistant leukemia model. Cotylenin A may be useful for differentiation therapy of retinoid-resistant leukemia.     

Sulforaphane sensitizes tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis through downregulation of ERK and Akt in lung adenocarcinoma A549 cells

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in some cancer cells, including A549 lung adenocarcinoma cells. However, treatment with TRAIL in combination with subtoxic concentrations of sulforaphane (SFN) sensitizes TRAIL-resistant A549 cells to TRAIL-mediated apoptosis. Combined treatment with SFN and TRAIL induced chromatin condensation, DNA fragmentation, annexin V staining and sub-G(1) phase DNA content. These indicators of apoptosis correlate with the induction of caspase-3 activity that results in the cleavage of poly(ADP-ribose) polymerase and the release of lactate dehydrogenase. Both the cytotoxic effect and apoptotic characteristics induced by combined treatment were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, demonstrating the important role of caspase-3 in the observed cytotoxic effect. Combined treatment also triggered the activation of p38 MAPK and JNK, and downregulation of ERK and Akt. Inhibitors of ERK (PD98059) or Akt (LY294002), but not p38 MAPK, resulted in significantly decreased cell viability. Although the activation of JNK was increased in response to combined treatment, inhibition of the JNK pathway significantly attenuated cell viability. These results indicate that caspase-3 is a key regulator of apoptosis in response to combined SFN and TRAIL in human lung adenocarcinoma A549 cells through downregulation of ERK and Akt.     

重组人TRAIL促卵巢癌细胞凋亡作用的研究

目的:分析重组人可溶性肿瘤坏死因子相关的凋亡诱导配体(TRAIL)分子诱导卵巢癌细胞的凋亡。方法:观察重组人可溶性TRAIL对卵巢癌细胞直接细胞毒作用,MTT法分析不同浓度TRAIL对卵巢癌细胞3AO、HO-8910的生长抑制率,采用流式细胞技术观察TRAIL作用于3AO后的凋亡率的变化。结果:重组人可溶性TRAIL蛋白可抑制卵巢癌细胞生长,诱导卵巢癌细胞发生凋亡;倒置显微镜下观察细胞呈现圆形固缩形态,细胞凋亡之后存活细胞减少。结论:重组人可溶性TRAIL具有明显诱导卵巢癌细胞凋亡的活性,并具有量效性及时效性。其抑制卵巢癌细胞机制可能与其促凋亡作用有关。     

The farnesyltransferase inhibitor R115777 up-regulates the expression of death receptor 5 and enhances TRAIL-induced apoptosis in human lung cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in transformed or malignant cells, thus exhibiting potential as a tumor-selective apoptosis-inducing cytokine for cancer treatment. Many studies have shown that the apoptosis-inducing activity of TRAIL can be enhanced by various cancer therapeutic agents. R115777 (tipifarnib) is the first farnesyltransferase inhibitor (FTI) that showed clinical activity in myeloid malignancies. In general, R115777, like other FTIs, exerts relatively weak effects on the induction of apoptosis in cancer cells with undefined mechanism(s). In the current study, we studied its effects on the growth of human lung cancer cells, including induction of apoptosis, and examined potential underlying mechanisms for these effects. We showed that R115777 induced apoptosis in human lung cancer cells, in addition to inducing G(1) or G(2)-M arrest. Moreover, we found that R115777 up-regulated the expression of death receptor 5 (DR5), an important death receptor for TRAIL, and exhibited an augmented effect on the induction of apoptosis when combined with recombinant TRAIL. Blockage of DR5 induction by small interfering RNA (siRNA) abrogated the ability of R115777 to enhance TRAIL-induced apoptosis, indicating that R115777 augments TRAIL-induced apoptosis through up-regulation of DR5 expression. Thus, our findings show the efficacy of R115777 in human lung cancer cells and suggest that R115777 may be used clinically in combination with TRAIL for treatment of human lung cancer.     

Cardiac glycosides initiate Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cells by up-regulation of death receptors 4 and 5

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the TNF family known to transduce their death signals via cell membrane receptors. Because it has been shown that Apo2L/TRAIL induces apoptosis in tumor cells without or little toxicity to normal cells, this cytokine became of special interest for cancer research. Unfortunately, cancer cells are often resistant to Apo2L/TRAIL-induced apoptosis; however, this can be at least partially negotiated by parallel treatment with other substances, such as chemotherapeutic agents. Here, we report that cardiac glycosides, which have been used for the treatment of cardiac failure for many years, sensitize lung cancer cells but not normal human peripheral blood mononuclear cells to Apo2L/TRAIL-induced apoptosis. Sensitization to Apo2L/TRAIL mediated by cardiac glycosides was accompanied by up-regulation of death receptors 4 (DR4) and 5 (DR5) on both RNA and protein levels. The use of small interfering RNA revealed that up-regulation of death receptors is essential for the demonstrated augmentation of apoptosis. Blocking of up-regulation of DR4 and DR5 alone significantly reduced cell death after combined treatment with cardiac glycosides and Apo2L/TRAIL. Combined silencing of DR4 and DR5 abrogated the ability of cardiac glycosides and Apo2L/TRAIL to induce apoptosis in an additive manner. To our knowledge, this is the first demonstration that glycosides up-regulate DR4 and DR5, thereby reverting the resistance of lung cancer cells to Apo2/TRAIL-induced apoptosis. Our data suggest that the combination of Apo2L/TRAIL and cardiac glycosides may be a new interesting anticancer treatment strategy.     

Transferrin ensures survival of ovarian carcinoma cells when apoptosis is induced by TNFalpha, FasL, TRAIL, or Myc

The activation of Myc induces apoptosis of human ovarian adenocarcinoma N.1 cells when serum factors are limited. However, the downstream mechanism that is triggered by Myc is unknown. Myc-activation and treatment with the proapoptotic ligands TNFalpha, FasL, and TRAIL induced H-ferritin expression under serum-deprived conditions. H-ferritin chelates intracellular iron and also intracellular iron sequestration by deferoxamine-induced apoptosis of N.1 cells. Supplementation of serum-free medium with holo-transferrin blocked apoptosis of N.1 cells that was induced by Myc-activation or by treatment with TNFalpha, FasL, and TRAIL, whereas apotransferrin did not prevent apoptosis. This suggests that intracellular iron depletion was a trigger for apoptosis and that transferrin-bound iron rescued N.1 cells. Furthermore, apoptosis of primary human ovarian carcinoma cells, which was induced by TNFalpha, FasL, and TRAIL, was also inhibited by holo-transferrin. The data suggest that Myc-activation, FasL, TNFalpha, and TRAIL disturbed cellular iron homeostasis, which triggered apoptosis of ovarian carcinoma cells and that transferrin iron ensured survival by re-establishing this homeostasis.     

Augmentation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by the synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) through up-regulation of TRAIL receptors in human lung cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis via the death receptors DR4 and DR5 in different transformed cells in vitro and exhibits potent antitumor activity in vivo with minor side effects. The synthetic retinoid CD437 is a potent inducer of apoptosis in cancer cells through increased levels of death receptors. We demonstrate that treatment of human lung cancer cells with a combination of suboptimal concentrations of CD437 and TRAIL enhanced induction of apoptosis in tumor cell lines with wild-type p53 but not in normal lung epithelial cells. CD437 up-regulated DR4 and DR5 expression. The CD437 and TRAIL combination enhanced activation of caspase-3, caspase-7, caspase-8, and caspase-9 and the subsequent cleavage of poly(ADP-ribose) polymerase and DNA fragmentation factor 45. Caspase inhibitors blocked the induction of apoptosis by this combination. Moreover, this combination induced Bid cleavage and increased cytochrome c release from mitochondria. These results suggest that the mechanism of enhanced apoptosis by this combination involves p53-dependent increase of death receptors by CD437, activation of these receptors by TRAIL, enhanced Bid cleavage, release of cytochrome c, and activation of caspase-3, caspase-7, caspase-8, and caspase-9. These findings suggest a novel strategy for the prevention and treatment of human lung cancer with the CD437 and TRAIL combination.     

Lipoxygenase inhibitors induce death receptor 5/TRAIL-R2 expression and sensitize malignant tumor cells to TRAIL-induced apoptosis

Lipoxygenases induce malignant tumor progression and lipoxygenase inhibitors have been considered as promising anti-tumor agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for new cancer therapeutics. Combined treatment with nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, and TRAIL markedly induced apoptosis in Jurkat T-cell leukemia cells at suboptimal concentrations for each agent. The combined treatment efficiently activated caspase-3, -8 and -10, and Bid. The underling mechanism by which NDGA enhanced TRAIL-induced apoptosis was examined. NDGA did not change the expression levels of anti-apoptotic factors, Bcl-x(L), Bcl-2, cIAP-1, XIAP and survivin. The expression of death receptor-related genes was investigated and it was found that NDGA specifically up-regulated the expression of death receptor 5 (DR5) at mRNA and protein levels. Down-regulation of DR5 by small interfering RNA prevented the sensitizing effect of NDGA on TRAIL-induced apoptosis. Furthermore, NDGA sensitized prostate cancer and colorectal cancer cells to TRAIL-induced apoptosis. In contrast, NDGA neither enhanced TRAIL-induced apoptosis nor up-regulated DR5 expression in normal peripheral blood mononuclear cells. Another lipoxygenase inhibitor, AA861, also up-regulated DR5 and sensitized Jurkat and DU145 cells to TRAIL. These results indicate that lipoxygenase inhibitors augment the apoptotic efficiency of TRAIL through DR5 up-regulation in malignant tumor cells, and raise the possibility that the combination of lipoxygenase inhibitor and TRAIL is a promising strategy for malignant tumor treatment.     

TRAIL对原代卵巢癌细胞增殖、凋亡及核 因子κB的影响

 目的 观察肿瘤坏死因子相关凋亡诱导配体（TNF-related apoptosis inducing- ligand; TRAIL）对原代卵巢癌细胞生长的抑制和诱导肿瘤细胞凋亡效应,以及细胞核因子κB(NF κＢ)激活和转运的影响。方法 来源于12例病人原代卵巢癌细胞,给予TRAIL 0、5、10、20、50、100μg/L分别作用24、48、72和96h,运用MTT比色法检测原代卵巢癌细胞生长、增殖情况;细胞免疫荧光法检测原代卵巢癌细胞凋亡和细胞内NF κB激活－转运情况。结果 TRAIL对原代卵巢癌细胞的生长抑制呈剂量依赖性,浓度大于20μg/L时抑制率增辐减慢;浓度相同时对癌细胞的抑制呈时间依赖性,72h后抑制率上升减缓;肿瘤细胞凋亡率随TRAIL作用时间延长而增加,5μg/L 24h、96h凋亡率分别是（28±1.18）%、（55±4.12）%;肿瘤细胞凋亡率也呈剂量依赖性;TRAIL作用后原代卵巢癌细胞质和胞核中NF κB表达增加。结论 TRAIL抑制原代卵巢癌细胞的增殖和生长,并诱导癌细胞的凋亡;激活癌细胞质中NF κB向细胞核内转运。     

High expression of crystallin αB represents an independent molecular marker for unfavourable ovarian cancer patient outcome and impairs TRAIL‐ and cisplatin‐induced apoptosis in human ovarian cancer cells

Dysregulated apoptotic pathways are regarded as major reasons for chemoresistance development as a particular challenge in ovarian cancer therapy. In search of molecular factors affecting human ovarian cancer cell apoptosis and, consequently, patient survival, we examined tumors of 103 platinum‐/taxane‐treated ovarian cancer patients by mRNA‐array hybridization, qPCR, and immunohistochemistry. We identified high expression of crystallin αB (CRYAB), a proposed negative regulator of tumor necrosis factor‐related apoptosis inducing ligand (TRAIL)‐mediated apoptosis. By Kaplan Meier analysis, this factor turned out to be significantly associated with poor patient outcome [overall survival (OS) p = 0.001, recurrence‐free survival (RFS) p = 0.003]. Elevated hazard ratios (HR) were estimated with regard to OS (HR = 2.11, 95% CI 1.10–4.06) and RFS (HR = 1.92, 95% CI 1.07–3.47) in multivariable analyses. These associations were confirmed in independent, publicly available mRNA data comprising 431 patients for OS (p < 0.001) and 413 for RFS (p < 0.001)_. Our findings were validated by studying apoptotic events in cultured human ovarian cancer cells which were stably transfected to express elevated CRYAB levels. These data emphasized the crucial role of CRYAB in human ovarian cancer biology since TRAIL‐ as well as cisplatin‐induced apoptosis was significantly impaired as a function of enhanced CRYAB expression. Taken together, we identified CRYAB as an independent biomarker for unfavourable outcome of human ovarian cancer patients. Since TRAIL is currently tested as anti‐cancer drug and large proportions of the present patient cohort displayed low CRYAB levels in their tumors, CRYAB may enable the selection of patient subgroups benefiting most from TRAIL‐containing therapy.     

The proteasome inhibitor PS-341 (bortezomib) up-regulates DR5 expression leading to induction of apoptosis and enhancement of TRAIL-induced apoptosis despite up-regulation of c-FLIP and survivin expression in human NSCLC cells

The proteasome inhibitor PS-341 (bortezomib or Velcade), an approved drug for treatment of patients with multiple myeloma, is currently being tested in clinical trials against various malignancies, including lung cancer. Preclinical studies have shown that PS-341 induces apoptosis and enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human cancer cells with undefined mechanisms. In the present study, we show that PS-341 induced caspase-8-dependent apoptosis, cooperated with TRAIL to induce apoptosis, and up-regulated death receptor 5 (DR5) expression in human non-small cell lung cancer (NSCLC) cells. DR5 induction correlated with the ability of PS-341 to induce apoptosis. Blockage of PS-341-induced DR5 up-regulation using DR5 small interfering RNA (siRNA) rendered cells less sensitive to apoptosis induced by either PS-341 or its combination with TRAIL, indicating that DR5 up-regulation mediates PS-341-induced apoptosis and enhancement of TRAIL-induced apoptosis in human NSCLC cells. We exclude the involvement of c-FLIP and survivin in mediating these events because c-FLIP (i.e., FLIP(S)) and survivin protein levels were actually elevated on exposure to PS-341. Reduction of c-FLIP with c-FLIP siRNA sensitized cells to PS-341-induced apoptosis, suggesting that c-FLIP elevation protects cells from PS-341-induced apoptosis. Thus, the present study highlights the important role of DR5 up-regulation in PS-341-induced apoptosis and enhancement of TRAIL-induced apoptosis in human NSCLC cells.