8-Cl-cAMP induces cell cycle-specific apoptosis in human cancer cells

8-Cl-cyclic adenosine monophosphate (8-Cl-cAMP) has been known to induce growth inhibition and differentiation in a variety of cancer cells by differential modulation of protein kinase A isozymes. To understand the anticancer activity of 8-Cl-cAMP further, we investigated the effect of 8-Cl-cAMP on apoptosis in human cancer cells. Most of the tested human cancer cells exhibited apoptosis upon treatment with 8-Cl-cAMP, albeit with different sensitivity. Among them, SH-SY5Y neuroblastoma cells and HL60 leukemic cells showed the most extensive apoptosis. The effect of 8-Cl-cAMP was not reproduced by other cAMP analogues or cAMP-elevating agents, showing that the effect of 8-Cl-cAMP was not caused by simple activation of protein kinase A (PKA). However, competition experiments showed that the binding of 8-Cl-cAMP to the cAMP receptor was essential for the induction of apoptosis. After the treatment of 8-Cl-cAMP, cells initially accumulated at the S and G2/M phases of the cell cycle and then apoptosis began to occur among the population of cells at the S/G2/M cell cycle phases, indicating that the 8-Cl-cAMP-induced apoptosis is closely related to cell cycle control. In support of this assumption, 8-Cl-cAMP-induced apoptosis was blocked by concomitant treatment with mimosine, which blocks the cell cycle at early S phase. Interestingly, 8-Cl-cAMP did not induce apoptosis in primary cultured normal cells and non-transformed cell lines, showing that 8-Cl-cAMP-induced apoptosis is specific to transformed cells. Taken together, our results show that the induction of apoptosis is one of the mechanisms through which 8-Cl-cAMP exerts anticancer activity.     

Polyporenic acid C induces caspase-8-mediated apoptosis in human lung cancer A549 cells

Lung cancer continues to be the leading cause of cancer-related mortality worldwide. This warrants the search for new and effective agents against lung cancer. We and others have recently shown that lanostane-type triterpenoids isolated from the fungal species Poria cocos (P. cocos) can inhibit cancer growth. However, the mechanisms responsible for the anticancer effects of these triterpenoids remain unclear. In this study, we investigated the effect of polyporenic acid C (PPAC), a lanostane-type triterpenoid from P. cocos, on the growth of A549 nonsmall cell lung cancer cells (NSCLC). The results demonstrate that PPAC significantly reduced cell proliferation via induction of apoptosis as evidenced by sub-G1 analysis, annexin V-FITC staining, and increase in cleavage of procaspase-8, -3, and poly(ADP-ribose)-polymerase (PARP). However, unlike our previously reported lanostane-type triterpenoid, pachymic acid, treatment of cells with PPAC was not accompanied by disruption of mitochondrial membrane potential and increase in cleavage of procaspase-9. Further, PPC-induced apoptosis was inhibited by caspase-8 and pan caspase inhibitors but not by a caspase-9 inhibitor. Taken together, the results suggest that PPAC induces apoptosis through the death receptor-mediated apoptotic pathway where the activation of caspase-8 leads to the direct cleavage of execution caspases without the involvement of the mitochondria. Furthermore, suppressed PI3-kinase/Akt signal pathway and enhanced p53 activation after PPAC treatment suggests this to be an additional mechanism for apoptosis induction. Together, these results encourage further studies of PPAC as a promising candidate for lung cancer therapy.     

XIAP regulates Akt activity and caspase-3-dependent cleavage during cisplatin-induced apoptosis in human ovarian epithelial cancer cells

Chemoresistance is a major hurdle for successful cancer therapy. Although multiple mechanisms have been implicated to be involved in cisplatin resistance, recent evidence has suggested that X-linked inhibitor of apoptosis protein (XIAP) may be a key determinant in chemosensitivity in ovarian cancer. Cell fate is determined by a balance between cell survival and apoptotic signaling. Whereas phosphatidylinositol 3-kinase (PI 3-K) and XIAP are believed to be important cell survival factors in human ovarian surface epithelial cancer cells, if and how they interact to confer resistance to chemotherapy is not known. In the present study, we have investigated the role of XIAP in the regulation of the PI 3-K/Akt survival pathway in chemosensitive (A2780-s, OV2008, and OVCAR-3) and resistant (A2780-cp) ovarian cancer cell lines and the nature of this interaction in cell death/survival signaling. Cisplatin decreased XIAP protein levels and induced Akt cleavage and apoptosis in chemosensitive, but not in resistant, ovarian cancer cells. Cisplatin also induced cleavage of caspase-9 and caspase-3, a process blocked by XIAP overexpression. Pretreatment of ovarian cancer cells and their whole cell lysate with tetrapeptide inhibitors of caspases in vitro significantly decreased Akt cleavage induced by cisplatin and exogenous active caspase-3. Adenoviral sense XIAP cDNA expression increased XIAP protein levels and increased Akt phosphorylation, indicative of activation of Akt and, likely, of PI 3-K. This was associated with a decrease in cisplatin-induced apoptosis. In a cell line (OVCAR-3) where basal phosphorylated Akt levels were high, XIAP overexpression failed to increase further the level of this phosphoprotein. XIAP down-regulation induced Akt cleavage and apoptosis, and treatment of whole cell lysate with human recombinant active caspase-3 resulted in a similar pattern of Akt cleavage. In the presence of the PI 3-K inhibitor (LY294002), XIAP overexpression failed to block cisplatin-induced apoptosis and to induce Akt phosphorylation, suggesting that the site of action of XIAP is upstream of Akt in this cell survival pathway. Taken together, the results indicate that XIAP prevents apoptosis through a PI 3-K-dependent inhibition of the caspase cascade. These results demonstrate a novel mechanism by which XIAP regulates apoptosis and the possible involvement of the PI 3-K/Akt survival pathway in XIAP-mediated chemoresistance of ovarian cancer cells.     

Synergistic antiproliferative effect of human recombinant interferons and retinoic acid in cultured breast cancer cells

A combination of retinoic acid (RA) and human recombinant DNA-derived interferon-gamma (Hu-IFN-gamma) was tested with respect to the growth inhibitory action on several human mammary carcinoma cell lines (ZR-75.1, 734-B, MCF-7, and BT-20), a human lung carcinoma cell line (CCL-185), and a human laryngeal carcinoma cell line (HEP-2). The mammary carcinoma cell lines were all sensitive to Hu-IFN-gamma, and 2 of them (ZR-75.1 and 734-B) were also affected by RA. The combination of both substances led to a pronounced synergistic amplification of growth inhibition in ZR-75.1 and 734-B cells. RA also increased the antiproliferative activity of Hu-IFN-gamma in the RA-resistant BT-20 cells and to a less pronounced degree in MCF-7 cells. In contrast to these findings, no synergistic effects were observed between Hu-IFN-gamma and RA in CCL-185 and HEP-2 cells. Human recombinant DNA-derived interferon-alpha 2 amplified the action of RA only in BT-20 cells, but it did not act synergistically with RA in the other cell lines tested.     

Cross talk between apoptosis and invasion signaling in cancer cells through caspase-3 activation

In solid tumors, cancer cells are exposed to various microenvironmental stresses such as hypoxia, nutritional depletion, and low pH. Cancer cells adapt to these stresses and circumvent cell death. When the antiapoptotic signals overcome the stress, cancer cells might acquire physiologic functions, such as invasiveness, instead of cell death. Here, we report that tumor cells acquire an invasive capacity from apoptotic signals through caspase activation. We treated rat ascites hepatoma MM1 cells with an apoptosis-inducing drug, etoposide, or hypoxia, and assessed the invasion capacity with an in vitro bioassay. Although MM1 cells hardly showed invasiveness in serum-free medium, under stress conditions, invasive capacity accompanied with morphologic change was induced with caspase-3 activation. Such stress-induced invasion as well as morphologic change was suppressed by blocking caspase-3 activity with caspase inhibitors or by RNA interference of caspase-3. In contrast, lysophosphatidic acid-induced invasiveness was not affected by caspase-3 inhibition. These results suggest that caspase-3 activation contributes to the stress-induced invasive capacity of these cancer cells.     

Arsenic trioxide induces apoptosis in human gastric cancer cells through up-regulation of p53 and activation of caspase-3

Arsenic trioxide (As(2)O(3)) can induce clinical remission in patients suffering from acute promyelocytic leukemia, through induction of apoptosis and activation of caspases. We investigated the potential use of As(2)O(3) in human gastric cancer and its possible mechanisms. Human gastric cancer cell lines AGS and MKN-28 were treated with various concentrations (0.1 to 100 microM) of As(2)O(3) for 24 to 72 hr. Apoptosis was determined by acridine orange staining, flow cytometry and DNA fragmentation. Protein levels of p53, p21(waf1/cip1), c-myc, bcl-2 and bax were detected by Western blotting. Effects of As(2)O(3) on caspase-3 protease activity, its protein concentration and cleavage of poly(ADP)-ribose polymerase (PARP) were also studied. As(2)O(3) inhibited cell growth and induced apoptosis in both cell lines, though AGS cells were more sensitive. As(2)O(3) induced apoptosis in AGS cells in a concentration- and time-dependent manner. Treatment resulted in a marked increase in p53 protein levels as early as 4 hr. Co-incubation with p53 anti-sense oligo-nucleotide suppressed As(2)O(3)-induced intracellular p53 over-expression and apoptosis. As(2)O(3) increased the activity of caspase-3, with appearance of its 17 kDa peptide fragment, and cleavage of PARP, with appearance of the 85 kDa cleavage product, both in parallel with the induction of apoptosis. Both the tripeptide caspase inhibitor zVAD-fmk and the specific caspase-3 inhibitor DEVD-fmk partially suppressed As(2)O(3)-induced caspase-3 activation and apoptosis. As(2)O(3) inhibits cell growth and induces apoptosis in gastric cancer cells, involving p53 over-expression and activation of caspase-3. The potential use of this compound in the treatment of gastric cancer is worth further investigation.     

Ursolic acid induces apoptosis through caspase-3 activation and cell cycle arrest in HaCat cells

Ursolic acid (UA) is a pentacyclic triterpene compound isolated from many kinds of medicinal plants and present in human diet. In this study, we investigated the pro-apoptotic effect of UA on HaCat derived keratinocyte cell line. Treatment with UA decreased the viability of HaCat cells in a concentration- and time-dependent manner. In addition, cell cycle analysis revealed that UA treated HaCat cells were blocked predominantly in G1 phase. Moreover, expression of p21WAF1, a cell cycle regulator, was increased by UA, indicating that UA-induced cell cycle arrest could be mediated through p21WAF1. During UA treatment, we also demonstrated that p53 was phosphorylated at serine 392 and translocated to the nucleus. It is well established that p53 achieves its tumor suppressor activity by inducing apoptosis on cells. To define the apoptotic process in our system, we examined effect of UA on caspase activities, and demonstrated caspase-3 activation. In conclusion, our results suggest that UA induces: i) cell cycle arrest concomitantly with the apparition of the apoptotic sub group G1 peak, and ii) cell death through apoptosis, which is mediated by caspase-3.     

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     

Synergistic effects of suberoylanilide hydroxamic acid combined with cisplatin causing cell cycle arrest independent apoptosis in platinum-resistant ovarian cancer cells

Histone deacetylase inhibitors (HDACIs) belong to an emerging class of anticancer compounds. It is increasingly recognized that their unique and complementary mode of action make HDACIs valuable agents in augmenting the cytotoxicity of conventional chemotherapeutics. We examined the potential for combined use of an approved HDACI, suberoylanilide hydroxamic acid (SAHA), with cisplatin (Cddp) in platinum-resistant ovarian cancer cells, OVCAR-3 and SKOV-3. The nature of the drug interaction following combinatory therapy was assessed using median effect analysis. We found that SAHA acted synergistically with Cddp over a wide range of concentrations in both cell types, resulting in favorable dose reductions of both compounds. In particular, in the more Cddp-resistant SKOV-3 cells, more than 8-fold dose reduction of Cddp was achieved with the simultaneous use of SAHA and Cddp as compared to the dose required to elicit similar cell kill effects using Cddp alone. More importantly, therapeutic selectivity for ovarian cancer cells over normal fibroblast cells were maintained with the combinatorial therapy. We further observed that the augmentation of Cddp-induced cell death was mediated by the net increase in apoptosis and was independent of cell cycle arrest. Overall, concurrent application of SAHA and Cddp yielded the most favorable cell kill, indicating that this combination is promising for treatment of platinum-resistant ovarian tumors.     

Effect of all-trans retinoic acid on telomerase activity in ovarian cancer cells

Vitamin A is an essential nutrient important for growth, vision, embryonic development, immune response and reproduction. Various retinoids have been shown to be effective chemotherapeutic and chemopreventive agents for a number of human cancers. Telomeres are nucleoprotein structures found at the end of chromosomes. During cellular division, the telomeres in normal cells shorten progressively and thus, function as a "molecular clock". Telomerase is a ribonucleoprotein complex that extends and maintains telomeres. Activation of telomerase is required for cells to overcome proliferative crisis. Telomerase activation is observed in 90% of human cancers, but not in normal somatic cells. We examined the role of telomerase in mediating the growth suppression of ovarian carcinoma cells by all-trans-retinoic acid (ATRA). Using a number of cell lines with varying levels of growth sensitivity to ATRA, we found that cells that exhibit ATRA-dependant suppression of growth also contained significantly reduced telomerase activity. We also observed a reduction in expression of the telomerase components, hTERT and hTR in ATRA treated ovarian carcinoma cells. Our results suggest that one mechanism by which ATRA acid inhibits cancer cell growth is by suppressing telomerase activity, thereby pushing cells to proliferative crisis.     

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.     

Hydroxycamptothecin-loaded Fe3O4 nanoparticles induce human lung cancer cell apoptosis through caspase-8 pathway activation and disrupt tight junctions

10-Hydroxycamptothecin (HCPT) elicits strong anti-cancer effects and is less toxic than camptothecin (CPT), making it widely used in recent clinical trials. However, its low solubility limits its application as an effective anti-cancer therapy. In the present study we investigate the hypothesis that the unique water dispersible oleic acid-Triton X-100-coated Fe3O4 nanoparticles loaded with HCPT disrupt epithelial cell-cell junctions and induce human lung cancer cell apoptosis through the caspase-8 pathway. We characterized the HCPT-loaded nanoparticles and determined their effects on lung cancer cell viability and apoptosis by using immunofluorescence light microscopy and SDS-PAGE/immunoblots. We found that HCPT-loaded nanoparticles elicited an anti-proliferative effect in a dose-dependent manner. HCPT-loaded nanoparticles reduced the expression of cell-cell junction protein claudins, E-cadherin and ZO-1, and transmission electron microcopy demonstrated a disrupted tight junction ultrastructure. Transepithelial electric resistance was also reduced, indicating the reduction of tight junction functions. The HCPT-loaded nanoparticles increased phosphorylation of p38 and SAPK/JNK while it showed no effects on p42/44 MAP kinase. Compared with void Fe3O4 nanoparticles or HCPT drug alone, HCPT drug-loaded nanoparticles evoked synergistic effects by increasing cell apoptosis with enhanced activation of the caspase-8 pathway. Therefore, our current study highlights the potential of HCPT drug-loaded nanoparticles as a chemotherapeutic agent for increasing anti-cancer drug efficacy.     

手霉素对人卵巢癌3AO细胞增殖和凋亡的影响

目的:探讨手霉素对人卵巢癌3AO细胞体外增殖和凋亡的影响及其可能的作用机制.方法:MTT法检测手霉素对3AO细胞的增殖抑制作用;Giemsa染色、透射电子显微镜下观察及FCM分析手霉素对3AO细胞的凋亡诱导作用;FCM检测手霉素对3AO细胞 ras、survivin和核因子-κB(nuclear factor-kappa B, NF-κB)(p65)蛋白表达的影响.结果:手霉素能显著抑制3AO细胞的增殖(P<0.05),并且这种抑制作用随着手霉素浓度的增加和作用时间的延长而增强.Giemsa染色和透射电子显微镜下观察发现,手霉素可诱导3AO细胞出现典型的凋亡形态学变化及超微结构改变.FCM分析结果显示,与对照组相比,手霉素诱导3AO细胞凋亡率明显增加(P<0.05),细胞周期中的G2/M期细胞也明显增加(P<0.05);3AO细胞的ras、NF-κB(p65)和survivin蛋白的表达水平明显下降(P<0.05).结论:手霉素可明显抑制人卵巢癌3AO细胞的增殖并诱导其凋亡,其作用机制可能与下调细胞ras、NF-κB(p65)和survivin蛋白的表达有关.     

Ursolic acid induces Bax-dependent apoptosis through the caspase-3 pathway in endometrial cancer SNG-II cells

The goal of this study was to examine the effect of ursolic acid, a pentacyclic triterpenoid compound, on growth of the endometrial cancer cell line SNG-II. We found that ursolic acid strongly inhibited the growth of SNG-II cells in a dose- and time-dependent manner. Morpholgical changes characteristic of apoptosis were observed in treated cells, such as the presence of apoptotic bodies and fragmentation of DNA into oligonucleosomal-sized fragments. We also investigated the active forms of caspase-3, -8 and -9 in ursolic acid-treated SNG-II cells. At 25 and 50 microM strength, ursolic acid induced marked increases in caspase-3 activity to approximately 5-fold that of control cells. Levels of cleaved caspase-3 increased in a time- and dose-dependent manner. Activation of caspases also led to the cleavage of target proteins, such as PARP. Ursolic acid treatment also resulted in a cleavage of poly (ADP-ribose) polymerase in a dose-dependent manner. Testing whether caspase-3 activation and DNA polymerase activity were inhibited by addition of Ac-DEDV-HCO during ursolic acid treatment showed that 50 microM Ac-DEDV-HCO inhibited caspase-3 activity in treated cells. Although DNA fragmentation was observed after ursolic acid treatment, DNA fragmentation did not occur in SNG II cells treated with both Ac-DEDV-HCO and ursolic acid. Because some researchers have suggested that mitochondrial pathways are involved in ursolic acid-induced apoptosis secondary to induction of mitochondrial cytochrome c release, we studied mitochondrial events in ursolic acid-induced apoptosis in these cell lines. After ursolic acid treatment, the anti-apoptotic Bcl-2 protein decreased and Bax expression was enhanced. Our results indicated that ursolic acid induced apoptotic processes in the endometrial cancer SNG-II cell line through mechanisms involving mitochondrial pathways and Bcl-2 family proteins.     

Chitosan induces apoptosis via caspase-3 activation in bladder tumor cells.

Recently, because of its low toxicity and biological effects, chitosan has been widely used in the medical and pharmaceutical fields, e.g., for nasal or oral delivery of peptide or polar drug delivery. Here, we report a growth-inhibitory effect of chitosan on tumor cells. The growth inhibition was examined by WST-1 colorimetric assay and cell counting. We also observed DNA fragmentation, which is characteristic of apoptosis, and elevated caspase-3-like activity in chitosan-treated cancer cells. The findings suggest that chitosan may have potential value in cancer therapy.     

Synergistic action of apoptosis induced by eicosapentaenoic acid and TNP-470 on human breast cancer cells.

The effects of eicosapentaenoic acid (EPA) and an angiogenesis inhibitor (TNP-470) on the suppression of breast cancer cell growth were examined in five human breast cancer cell lines (MDA-MB-231, T-47D, MCF-7, KPL-1, and MKL-F). In all five cell lines, EPA and TNP-470 alone both showed tumor growth inhibition in a time- and dose-dependent manner, and in combination, a synergistic effect was seen at high concentrations. EPA plus TNP-470 treatment evoked apoptosis as confirmed by the appearance of sub G1 populations, by DNA fragmentation, and by cell morphology. With the combination, the expression of Bax and Bcl-xS, the apoptosis-enhancing proteins, was more up-regulated and that of Bcl-2 and Bcl-xL, the apoptosis-suppressing proteins, was more down-regulated compared to the use of EPA or TNP-470 alone, suggesting that their synergistic effect was due to an acceleration of apoptosis.     

Ursolic acid induces apoptosis through mitochondrial intrinsic pathway and caspase-3 activation in M4Beu melanoma cells

Over the coming years, skin cancer could become a significant public health problem. Previous results indicate that ursolic acid (UA), a pentacyclic triterpene acid, has pleiotropic biologic activities such as antiinflammatory and antiproliferative activities on cancer cells. As UA represents a promising chemical entity for the protection of human skin, in agreement with tests done by the cosmetic industry, we investigated its effects on the M4Beu human melanoma cell line. In this report, we demonstrated for the first time that UA had a significant antiproliferative effect on M4Beu, associated with the induction of an apoptotic process, characterized by caspase-3 activation, the downstream central effector of apoptosis. We demonstrated that UA-induced apoptosis was dependent on the mitochondrial intrinsic pathway, as shown by transmembrane potential collapse (DeltaPsim) and by alteration of the Bax-Bcl-2 balance, with a concomitant increase in Bax expression and decrease in Bcl-2 expression. We also showed that UA-induced DeltaPsim was associated with apoptosis-inducing factor leakage from mitochondria. Taken together, our results suggest that UA-induced apoptosis on M4Beu cells is accomplished via triggering of mitochondrial pathway. In conclusion, UA could be an encouraging compound in the treatment or prevention of skin cancer and may represent a new promising anticancer agent in the treatment of melanoma.