Addition of anticancer agents enhances freezing-induced prostate cancer cell death: implications of mitochondrial involvement

Recent evidence suggests that the successful treatment of prostate cancer may require adjuvant therapies. Accordingly, a better understanding of the molecular mechanisms involved in current treatments may lead to enhanced efficacy by providing a basis for adjuvant therapies. In this study, we demonstrate that the combination of sub-lethal concentrations of chemotherapeutic agents prior to freezing (-15 degrees C) in a prostate cancer cell (PC-3) model results in enhanced efficacy over either treatment alone. Morphological analysis revealed that necrosis appeared to be the prevalent mode of cell death following adjuvant (in vitro) modeling, yet molecular analysis indicated that freezing and chemotherapy differentially activated apoptotic cascades through modulating opposing members of the Bcl-2 protein family. Freezing results in a time-dependent increase of the antiapoptotic Bcl-2 protein, while chemotherapy results in an increase of the pro-apoptotic Bax protein. Anti-apoptotic Bcl-2 protein levels increase over 3-fold following exposure to freezing. 5-Fluorouracil (5-FU) causes pro-apoptotic Bax levels to increase 2-fold during the drug exposure. The increase in Bax was also apparent following the combination of 5-FU/freezing, while Bcl-2 levels were maintained at or below control levels. This led to a shift in the Bcl-2 to Bax ratio to a pro-death tendency. Other effective cryo/chemo combinations were also found to provide similar effects. The combination of cisplatin/freezing resulted in a 4-fold increase in the ratio of Bax to Bcl-2 when compared to controls, which represented a 2-fold increase over the 5-FU/freezing-combination model. This increase may contribute to the continued reduction in cell number observed during the 13-day recovery period. Additionally, the addition of an apoptotic caspase inhibitor was not able to protect cultures from cell death following combination treatment. In conclusion, the data suggest that both Bcl-2 and Bax may, not only, play an important role in the efficacy of the cryo/chemo combination, but also the balance between the two may determine the role and extent of system destruction.     

Phytaspases: Aspartate-specific proteases involved in plant cell death

Structures and properties of the recently found aspartate-specific cell death-related plant proteases called phytaspases are reviewed and compared to those of caspases, animal apoptotic proteases. Caspases (cysteine-dependent proteases) and phytaspases (serine-dependent proteases) dramatically differ in structure, although manifest a similar substrate specificity and a play a similar role in the programmed cell death. Distinctions in the structural organization of animal and plant death proteases were shown to define differences in the regulation strategies of functioning of these proteolytic enzymes in the two kingdoms.     

Transglutaminase activity is involved in polyamine-induced programmed cell death.

Natural polyamines, i.e., putrescine, spermidine, and spermine, are ubiquitous molecules essential for cell proliferation and differentiation. In the present study, the effect of polyamines on primary cultures of bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and a human melanoma cell line was examined. While in the absence of fetal calf serum (FCS) polyamines had no effect on viability, in the presence of FCS spermidine and spermine, at concentrations close to physiologic levels, induced a dose-dependent cell death, whereas putrescine was ineffective. RASMCs were significantly more sensitive than other cells. FACS analysis, oligo-nucleosome ELISA, Hoechst nuclear staining, and Annexin V-FITC quantification showed that cell death was likely due to apoptosis. Cells exposed to spermidine showed a marked increase of intracellular transglutaminase (TGase) activity ( approximately 30-fold over control). Inhibitors of polyamine oxidation or inhibitors of TGase activity prevented polyamine-induced apoptosis. Moreover, tissue TGase overexpression significantly increased cell sensitivity to polyamine, suggesting that this effect is likely related to enhanced intracellular TGase activity. These data indicate that polyamines may modulate cell viability through a novel TGase-dependent process.     

Resilience of death: intrinsic disorder in proteins involved in the programmed cell death

It is recognized now that intrinsically disordered proteins (IDPs), which do not have unique 3D structures as a whole or in noticeable parts, constitute a significant fraction of any given proteome. IDPs are characterized by an astonishing structural and functional diversity that defines their ability to be universal regulators of various cellular pathways. Programmed cell death (PCD) is one of the most intricate cellular processes where the cell uses specialized cellular machinery and intracellular programs to kill itself. This cell-suicide mechanism enables metazoans to control cell numbers and to eliminate cells that threaten the animal''s survival. PCD includes several specific modules, such as apoptosis, autophagy, and programmed necrosis (necroptosis). These modules are not only tightly regulated but also intimately interconnected and are jointly controlled via a complex set of protein–protein interactions. To understand the role of the intrinsic disorder in controlling and regulating the PCD, several large sets of PCD-related proteins across 28 species were analyzed using a wide array of modern bioinformatics tools. This study indicates that the intrinsic disorder phenomenon has to be taken into consideration to generate a complete picture of the interconnected processes, pathways, and modules that determine the essence of the PCD. We demonstrate that proteins involved in regulation and execution of PCD possess substantial amount of intrinsic disorder. We annotate functional roles of disorder across and within apoptosis, autophagy, and necroptosis processes. Disordered regions are shown to be implemented in a number of crucial functions, such as protein–protein interactions, interactions with other partners including nucleic acids and other ligands, are enriched in post-translational modification sites, and are characterized by specific evolutionary patterns. We mapped the disorder into an integrated network of PCD pathways and into the interactomes of selected proteins that are involved in the p53-mediated apoptotic signaling pathway.     

Protein aggregates: an aging factor involved in cell death

In a previous study, we demonstrated the presence of protein aggregates in an exponentially grown Escherichia coli culture. In light of these observations, protein aggregates could be considered damage to cells that is able to pass from one generation to the next. Based on the assumption that the amount of aggregate protein could represent an aging factor, we monitored this amount in a bacterial culture during senescence. In doing so, we observed (i) a significant increase in the amount of aggregate protein over time, (ii) a proportional relationship between the amount of aggregate protein and the level of dead cells, (iii) a larger amount in dead cells than in culturable cells, (iv) a heterogeneous distribution of different amounts within a homogenous population of culturable cells entering stasis, and (v) that the initial amount of aggregate protein within a culturable population conditioned the death rate of the culture. Together, the results presented in this study suggest that protein aggregates indeed represent one aging factor leading to bacterial cell death.     

Another DRAM involved in autophagy and cell death

Macroautophagy (hereafter referred to as autophagy) is controlled by a number of core proteins that are critical for all autophagy responses. In addition, a number of autophagy regulators have been found that are not critical for macroautophagy per se, but which play roles in regulating autophagy in either selective situations or in response to specific stimuli. In a recent study, we reported the initial characterization of a new autophagy regulator encoded by TMEM150B that is related to the Damage-Regulated Autophagy Modulator, DRAM1. We have termed this factor DRAM3 for DRAM-Related/Associated Member 3. Interestingly, like DRAM1, DRAM3 regulates both autophagy and cell death, but we found these two functions of the protein are not intrinsically connected.     

Pathways involved in sunburn cell formation: deregulation in skin cancer.

The incidence of squamous cell carcinoma of the skin is rising worldwide for decades. Chronic exposure to sunlight is the most important environmental risk factor for this type of skin cancer. This is predominantly due to the DNA damaging effect of ultraviolet-B (UVB) in sunlight. UVB induces also sunburn cells, i.e. apoptotic keratinocytes, which is a crucial protective mechanism against the carcinogenic effects of UVB irradiation. This process is regulated by a wide range of molecular determinants involved in the balance between pro- and anti-apoptotic pathways. Growing evidence suggests that the deregulation of this balance by chronic UVB irradiation, contributes to the development of skin cancer. This review gives a brief summary of major known pathways involved in the regulation of keratinocyte survival and cell death upon UVB damage and discusses the contribution of the deregulation of these cascades to photocarcinogenesis.     

Endo-exonucleases: Enzymes involved in DNA repair and cell death?

Endo-exonucleases from E. coli to man, although very different proteins, are multifunctional enzymes with similar enzymatic activities. They probably have two common but opposing biological roles. On the one hand, they promote survival of the organism by acting in recombination and recombinational DNA repair to diversify and help preserve the genome intact. On the other hand, they degrade the genomic DNA when it is damaged beyond repair. This ensures elimination of heavily mutagenized cells from the population.     

MAPK signaling is involved in camptothecin-induced cell death

Camptothecin, a topoisomerase I inhibitor, is a well-known anticancer drug. However, its mechanism has not been well studied in human gastric cancer cell lines. Camptothecin induced apoptotic cell death in human gastric cancer cell line AGS. Z-VAD-fmk, pan-caspase inhibitor, blocked apoptotic phenotypes induced by camptothecin suggesting that caspases are involved in camptothecin-induced cell death. An inhibitor of caspase-6 or -8 or -9 did not prevent cell death by camptothecin. Various protease inhibitors failed to prevent camptothecin-induced cell death. These results suggest that only few caspases are involved in camptothecin-induced cell death. Camptothecin induced phosphorylation of ERK1/2, JNK, and p38 MAPK, in a dose and time-dependent manner in AGS. Z-VAD-fmk did not affect MAPK signaling induced by camptothecin suggesting that caspase signaling occurs downstream of MAPK signaling. Blocking of p38 MAPK, but not ERK1/2, resulted in partial inhibition of cell death and PARP cleavage by camptothecin in AGS. Taken together, MAPK signaling is associated with apoptotic cell death by camptothecin.     

Escaping cell death: survival proteins in cancer

Defects in apoptosis signaling pathways are common in cancer cells. Such defects may play an important role in tumor initiation because apoptosis normally eliminates cells with damaged DNA or dysregulated cell cycle, i.e., cells with increased malignant potential. Moreover, impaired apoptosis may enhance tumor progression and promote metastasis by enabling tumor cells to survive the transit in the bloodstream and to grow in ectopic tissue sites lacking the otherwise required survival factors. Finally, raised apoptosis threshold may have deleterious consequences by rendering cancer cells resistant to various forms of therapy. The intensive apoptosis research during the past decade has resulted in the identification of several proteins which may promote tumorigenesis by inhibiting apoptosis. Of special relevance in human cancer are those commonly expressed in primary tumors and functioning at the common part of the signaling pathway leading to apoptosis. Proteins fulfilling these criteria include antiapoptotic members of the Bcl-2 protein family, heat shock proteins, Hsp70 and Hsp27, as well as survivin, the novel cancer-associated member of the inhibitor of apoptosis protein family. Understanding the molecular mechanisms of action of these proteins may offer novel modes of rationally and selectively manipulating the sensitivity of cancer cells to therapy.     

BCL-2 family proteins: Regulators of cell death involved in the pathogenesis of cancer and resistance to therapy

The BCL-2 gene was first discovered because of its involvement in the t(14;18) chromosomal translocations commonly found in lymphomas, which result in deregulation of BCL-2 gene expression and cause inappropriately high levels of Bcl-2 protein production. Expression of the BCL-2 gene can also become altered in human cancers through other mechanisms, including loss of the p53 tumor suppressor which normally functions as a repressor of BCL-2 gene expression in some tissues. Bcl-2 is a blocker of programmed cell death and apoptosis that contributes to neoplastic cell expansion by preventing cell turnover caused by physiological cell death mechanisms, as opposed to accelerating rates of cell division. Overproduction of the Bcl-2 protein also prevents cell death induced by nearly all cytotoxic anticancer drugs and radiation, thus contributing to treatment failures in patients with some types of cancer. Several homologs of Bcl-2 have recently been discovered, some of which function as inhibitors of cell death and others as promoters of apoptosis that oppose the actions of the Bcl-2 protein. Many of these Bcl-2 family proteins can interact through formation of homo- and heterotypic dimers. In addition, several nonhomologous proteins have been identified that bind to Bcl-2 and that can modulate apoptosis. These protein-protein interactions may eventual serve as targets for pharmacologically manipulating the physiological cell death pathway for treatment of cancer and several other diseases. © 1996 Wiley-Liss, Inc.     

Cryoablation and immunotherapy of cancer

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MDA-7/IL-24 induces Bcl-2 denitrosylation and ubiquitin-degradation involved in cancer cell apoptosis

MDA-7/IL-24 was involved in the specific cancer apoptosis through suppression of Bcl-2 expression, which is a key apoptosis regulatory protein of the mitochondrial death pathway. However, the underlying mechanisms of this regulation are unclear. We report here that tumor-selective replicating adenovirus ZD55-IL-24 leads to Bcl-2 S-denitrosylation and concomitant ubiquitination, which take part in the 26S proteasome degradation. IL-24-siRNA completely blocks Bcl-2 ubiquitination via reversion of Bcl-2 S-denitrosylation and protects it from proteasomal degradation which confirmed the significant role of MDA-7/IL-24 in regulating posttranslational modification of Bcl-2 in cancer cells. Nitric oxide (NO) is a key regulator of protein S-nitrosylation and denitrosylation. The NO donor, sodium nitroprusside (SNP), down-regulates Bcl-2 S-denitrosylation, attenuates Bcl-2 ubiquitination and subsequently counteracts MDA-7/IL-24 induced cancer cell apoptosis, whereas NO inhibitor 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (PTIO) shows the opposite effect. At the same time, these NO modulators fail to affect Bcl-2 phosphorylation, suggesting that NO regulates Bcl-2 stability in a phosphorylation-independent manner. In addition, Bcl-2 S-nitrosylation reduction induced by ZD55-IL-24 was attributed to both iNOS decrease and TrxR1 increase. iNOS-siRNA facilitates Bcl-2 S-denitrosylation and ubiquitin-degradation, whereas the TrxR1 inhibitor auranofin prevents Bcl-2 from denitrosylation and ubiquitination, thus restrains the caspase signal pathway activation and subsequent cancer cell apoptosis. Taken together, our studies reveal that MDA-7/IL-24 induces Bcl-2 S-denitrosylation via regulation of iNOS and TrxR1. Moreover, denitrosylation of Bcl-2 results in its ubiquitination and subsequent caspase protease family activation, as a consequence, apoptosis susceptibility. These findings provide a novel insight into MDA-7/IL-24 induced growth inhibition and carcinoma apoptosis.     

果蝇蜕皮激素诱导程序性细胞死亡的遗传调控因子

近年来关于果蝇程序性细胞死亡（programmed cell death, PCD）的研究结果表明,在果蝇的变态发育过程中,蜕皮激素与受体结合后诱导转录因子的表达。这些转录因子作为程序性细胞死亡调控网络中的初、次级应答信号,激活凋亡诱导因子Reaper、Hid和Grim的表达。Reaper、Hid和Grim进而阻止凋亡蛋白抑制因子的活性,从而启动半胱氨酸蛋白酶caspase途径,引起细胞凋亡（apoptosis）。该文综述了蜕皮激素诱导的果蝇程序性细胞死亡中各遗传调控因子之间的关系。     

Molecules involved in the modulation of rapid cell death in Xanthomonas

In earlier studies from this laboratory, Xanthomonas campestris pv. glycines was found to exhibit a nutrition stress-related postexponential rapid cell death (RCD). The RCD was exhibited in protein-rich media but not in starch or other minimal media. This RCD in X. campestris pv. glycines was found to display features similar to those of the programmed cell death (PCD) of eukaryotes. Results of the present study showed that the observed RCD in this organism is both positively and negatively regulated by small molecules. The amino acids glycine and l-alanine as well as the D isomers of valine, methionine, and threonine were found to induce the synthesis of an active caspase-3-like protein that was associated with the onset of RCD. Addition of pyruvate and citrate to the culture medium induced both the synthesis of active caspase-3-like protein and RCD. Higher levels of intracellular accumulation of pyruvate and citrate were also observed under conditions favoring RCD. On the other hand, dextrin and maltose, the hydrolytic products of starch, inhibited the synthesis of the caspase-3-like protein. Addition of glucose and cyclic AMP (cAMP) to the RCD-favoring medium prevented RCD. Glucose, cAMP, caffeine (a known inhibitor of a phosphodiesterase that breaks down cAMP), and forskolin (from the herb Coleus forskholii, known to activate the enzyme adenylate cyclase that forms cAMP) inhibited the caspase enzyme activity in vivo and consequently the RCD process. The addition of glucose and other inhibitors of RCD enhanced intracellular cAMP accumulation. This is the first report demonstrating the involvement of small molecules in the regulation of nutrition stress-related stationary-phase rapid cell death in X. campestris pv. glycines, which is programmed.     

AMPD3 is involved in anthrax LeTx-induced macrophage cell death

The responses of macrophages to Bacillus anthracis infection are important for the survival of the host, since macrophages are required for the germination of B. anthracis spores in lymph nodes, and macrophage death exacerbates anthrax lethal toxin (LeTx)-induced organ collapse. To elucidate the mechanism of macrophage cell death induced by LeTx, we performed a genetic screen to search for genes associated with LeTx-induced macrophage cell death. RAW 264.7 cells, a macrophage-like cell line sensitive to LeTx-induced death, were randomly mutated and LeTx-resistant mutant clones were selected. AMP deaminase 3 (AMPD3), an enzyme that converts AMP to IMP, was identified to be mutated in one of the resistant clones. The requirement of AMPD3 in LeTx-induced cell death of RAW 264.7 cells was confirmed by the restoration of LeTx sensitivity with ectopic reconstitution of AMPD3 expression. AMPD3 deficiency does not affect LeTx entering cells and the cleavage of mitogen-activated protein kinase kinase (MKK) by lethal factor inside cells, but does impair an unknown downstream event that is linked to cell death. Our data provides new information regarding LeTx-induced macrophage death and suggests that there is a key regulatory site downstream of or parallel to MKK cleavage that controls the cell death in LeTx-treated macrophages.     

Beta4 integrin is involved in statin-induced endothelial cell death

HMG-CoA reductase inhibitors (statins) have been shown to inhibit angiogenesis. The molecular mechanism mediating the anti-endothelial activities of statins remains unclear. The present study demonstrated that the antiangiogenic effect of atorvastatin (ATV) was associated with endothelial death. Molecular profiling data identified a 29-fold upregulation of beta4 integrin mRNA. Western blot and flow cytometry confirmed robust increases of total and cell-surface beta4 integrin. Blockage of beta4 integrin activity by antagonizing antibody abrogated ATV-induced endothelial death. The endothelial death and beta4 integrin upregulation by ATV could be reversed by intermediate metabilites of the HMG-CoA reductase pathway mevalonate or GGPP, but not by FPP, suggesting that these effects were results of specific inhibition of the pathway. These data indicate that the HMG-CoA reductase might represent an important survival pathway in angiogenic endothelial cells and thus, a potential target for antiangiogenic therapy.     

程序性细胞死亡与肿瘤

程序性细胞死亡（programmed cell death,PCD）是指由基因控制的细胞自主的有序性死亡方式,涉及一系列基因的激活、表达以及调控等。目前,经典细胞凋亡被称为Ⅰ型PCD,而自噬性细胞死亡称为Ⅱ型PCD,坏死样程序性细胞死亡则被称为Ⅲ型PCD,它们在肿瘤的发生、发展及治疗过程中起非常重要的作用。该文结合国内外最新研究进展主要针对不同细胞死亡模式及其相互作用、关键作用蛋白,细胞自噬与肿瘤发生,细胞自噬、凋亡与肿瘤治疗作一简要综述,并展望发展前景,提出在肿瘤治疗中如何利用不同死亡模式的协同作用最大限度地发挥其临床应用价值。     

Intracellular calcium chelators and oxidant-induced renal proximal tubule cell death.

The effect of intracellular calcium chelators on rabbit renal proximal tubule (RPT) cell death induced by t-butyl hydroperoxide (TBHP) and H2O2 was examined. Preincubation of RPT suspensions with 50 microM QUIN 2/AM completely prevented TBHP (0.5 mM) and H2O2 (2 mM) induced cell death [i.e., release of lactate dehydrogenase (LDH)]. QUIN 2/AM, BAPTA/AM, EGTA/AM, and FURA 2/AM, at 5 microM, decreased LDH release (at 6 hr) from 41% to 4%, 21%, 26%, and 33%, and decreased lipid peroxidation (at 1 hr) from 1.0 to 0.1, 0.4, 0.6, and 0.8 nmol MDA/mg protein, respectively, after TBHP exposure. Since oxidant-induced lipid peroxidation and cell death are iron-dependent in this model, these results suggest that the intracellular calcium chelators inhibit cell death by chelating iron.     

How cell death shapes cancer

Apoptosis has been established as a mechanism of anti-cancer defense. Members of the BCL-2 family are critical mediators of apoptotic cell death in health and disease, often found to be deregulated in cancer and believed to lead to the survival of malignant clones. However, over the years, a number of studies pointed out that a model in which cell death resistance unambiguously acts as a barrier against malignant disease might be too simple. This is based on paradoxical observations made in tumor patients as well as mouse models indicating that apoptosis can indeed drive tumor formation, at least under certain circumstances. One possible explanation for this phenomenon is that apoptosis can promote proliferation critically needed to compensate for cell loss, for example, upon therapy, and to restore tissue homeostasis. However, this, at the same time, can promote tumor development by allowing expansion of selected clones. Usually, tissue resident stem/progenitor cells are a major source for repopulation, some of them potentially carrying (age-, injury- or therapy-induced) genetic aberrations deleterious for the host. Thereby, apoptosis might drive genomic instability by facilitating the emergence of pathologic clones during phases of proliferation and subsequent replication stress-associated DNA damage. Tumorigenesis initiated by repeated cell attrition and repopulation, as confirmed in different genetic models, has parallels in human cancers, exemplified in therapy-induced secondary malignancies and myelodysplastic syndromes in patients with congenital bone marrow failure syndromes. Here, we aim to review evidence in support of the oncogenic role of stress-induced apoptosis.