Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms

Butyrate has antitumorigenic effects on colon cancer cells, inhibits cell growth and promotes differentiation and apoptosis. These effects depend on its intracellular concentration, which is regulated by its transport. We have analysed butyrate uptake kinetics in human colon adenocarcinoma cells sensitive to the apoptotic effects of butyrate (BCS-TC2, Caco-2 and HT-29), in butyrate-resistant cells (BCS-TC2.BR2) and in normal colonic cells (FHC). The properties of transport were analysed with structural analogues, specific inhibitors and different bicarbonate and sodium concentrations. Two carrier-mediated mechanisms were detected: a low-affinity/high-capacity (K(m)=109+/-16 mM in BCS-TC2 cells) anion exchanger and a high-affinity/low-capacity (K(m)=17.9+/-4.0 microM in BCS-TC2 cells) proton-monocarboxylate co-transporter that was energy-dependent and activated via PKCdelta (protein kinase Cdelta). All adenocarcinoma cells analysed express MCT (monocarboxylate transporter) 1, MCT4, ancillary protein CD147 and AE2 (anion exchanger 2). Silencing experiments show that MCT1, whose expression increases with butyrate treatment in butyrate-sensitive cells, plays a key role in high-affinity transport. Low-affinity uptake was mediated by a butyrate/bicarbonate antiporter along with a possible contribution of AE2 and MCT4. Butyrate treatment increased uptake in a time- and dose-dependent manner in butyrate-sensitive but not in butyrate-resistant cells. The two butyrate-uptake activities in human colon adenocarcinoma cells enable butyrate transport at different physiological conditions to maintain cell functionality. The high-affinity/low-capacity transport functions under low butyrate concentrations and may be relevant for the survival of carcinoma cells in tumour regions with low glucose and butyrate availability as well as for the normal physiology of colonocytes.     

Deoxycholic and chenodeoxycholic bile acids induce apoptosis via oxidative stress in human colon adenocarcinoma cells

The continuous exposure of the colonic epithelium to high concentrations of bile acids may exert cytotoxic effects and has been related to pathogenesis of colon cancer. A better knowledge of the mechanisms by which bile acids induce toxicity is still required and may be useful for the development of new therapeutic strategies. We have studied the effect of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) treatments in BCS-TC2 human colon adenocarcinoma cells. Both bile acids promote cell death, being this effect higher for CDCA. Apoptosis is detected after 30 min–2 h of treatment, as observed by cell detachment, loss of membrane asymmetry, internucleosomal DNA degradation, appearance of mitochondrial transition permeability (MPT), and caspase and Bax activation. At longer treatment times, apoptosis is followed in vitro by secondary necrosis due to impaired mitochondrial activity and ATP depletion. Bile acid-induced apoptosis is a result of oxidative stress with increased ROS generation mainly by activation of plasma membrane enzymes, such as NAD(P)H oxidases and, to a lower extent, PLA₂. These effects lead to a loss of mitochondrial potential and release of pro-apoptotic factors to the cytosol, which is confirmed by activation of caspase-9 and -3, but not caspase-8. This initial apoptotic steps promote cleavage of Bcl-2, allowing Bax activation and formation of additional pores in the mitochondrial membrane that amplify the apoptotic signal.     

Mitochondrial genome depletion in human liver cells abolishes bile acid-induced apoptosis: Role of the Akt/mTOR survival pathway and Bcl-2 family proteins

Acute accumulation of bile acids in hepatocytes may cause cell death. However, during long-term exposure due to prolonged cholestasis, hepatocytes may develop a certain degree of chemoresistance to these compounds. Because mitochondrial adaptation to persistent oxidative stress may be involved in this process, here we have investigated the effects of complete mitochondrial genome depletion on the response to bile acid-induced hepatocellular injury. A subline (Rho) of human hepatoma SK-Hep-1 cells totally depleted of mitochondrial DNA (mtDNA) was obtained, and bile acid-induced concentration-dependent activation of apoptosis/necrosis and survival signaling pathways was studied. In the absence of changes in intracellular ATP content, Rho cells were highly resistant to bile acid-induced apoptosis and partially resistant to bile acid-induced necrosis. In Rho cells, both basal and bile acid-induced generation of reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, was decreased. Bile acid-induced proapoptotic signals were also decreased, as evidenced by a reduction in the expression ratios Bax-α/Bcl-2, Bcl-xS/Bcl-2, and Bcl-xS/Bcl-xL. This was mainly due to a downregulation of Bax-α and Bcl-xS. Moreover, in these cells the Akt/mTOR pathway was constitutively activated in a ROS-independent manner and remained similarly activated in the presence of bile acid treatment. In contrast, ERK1/2 activation was constitutively reduced and was not activated by incubation with bile acids. In conclusion, these results suggest that impaired mitochondrial function associated with mtDNA alterations, which may occur in liver cells during prolonged cholestasis, may activate mechanisms of cell survival accounting for an enhanced resistance of hepatocytes to bile acid-induced apoptosis.     

Apoptosis and anticancer drug resistance.

Anticancer agents induce cancer cell death through apoptosis or necrosis. As anticancer agents at low and high concentrations cause apoptosis and necrosis, respectively, cancer cells may be merely injured by an anticancer agent in apoptosis, and cell death may result from an activation of the internal constituents to induce apoptosis. Therefore, an alternation of apoptotic pathway must change the efficacy of anticancer agents. As an escape of cancer cells from apoptosis seems to be closely associated with the development of anticancer resistance, this report focuses on mechanisms of apoptosis and its association with anticancer resistance. A Bax induces apoptosis mitochondria-dependently, whereas Fas can induce apoptosis mitochondria-independently. An interaction of Bax and Bcl-2 is very important to decide cell life or death, and Bcl-2 phosphorylation may control this interaction: Paclitaxel treatment induced Bcl-2 phosphorylation and typical apoptosis, whereas hyperthermia induced not Bcl-2 phosphorylation but nuclear translocation and failed to induce apoptosis. Moreover, Fas was localized in the cytoplasm of exponentially growing cells and on the cell membrane of confluent cells. We would like to emphasize that it is very important to check the localization of constituents of apoptosis in order to evaluate the susceptibility of cancer cells to apoptosis.     

Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes

Muscle cell apoptosis accompanies normal muscle development and regeneration, as well as degenerative diseases and aging. C2C12 murine myoblast cells represent a common model to study muscle differentiation. Though it was already shown that myogenic differentiation of C2C12 cells is accompanied by enhanced apoptosis in a fraction of cells, either the cell population sensitive to apoptosis or regulatory mechanisms for the apoptotic response are unclear so far. In the current study we characterize apoptotic phenotypes of different types of C2C12 cells at all stages of differentiation, and report here that myotubes of differentiated C2C12 cells with low levels of anti-apoptotic Bcl-2 expression are particularly vulnerable to apoptosis even though they are displaying low levels of pro-apoptotic proteins Bax, Bak and Bad. In contrast, reserve cells exhibit higher levels of Bcl-2 and high resistance to apoptosis. The transfection of proliferating myoblasts with Bcl-2 prior to differentiation did not protect against spontaneous apoptosis accompanying differentiation of C2C12 cells but led to Bcl-2 overexpression in myotubes and to significant protection from apoptotic cell loss caused by exposure to hydrogen peroxide. Overall, our data advocate for a Bcl-2-dependent mechanism of apoptosis in differentiated muscle cells. However, downstream processes for spontaneous and hydrogen peroxide induced apoptosis are not completely similar. Apoptosis in differentiating myoblasts and myotubes is regulated not through interaction of Bcl-2 with pro-apoptotic Bcl-2 family proteins such as Bax, Bak, and Bad.     

The antiapoptotic role of pregnane X receptor in human colon cancer cells

The orphan nuclear receptor pregnane X receptor (PXR) plays an important role in the detoxification of foreign and endogenous chemicals, including bile acids. PXR promotes bile acid elimination by activating bile acid-detoxifying enzymes and transporters. Certain bile acids are known to promote colonic carcinogenesis by inducing colon cancer cell apoptosis. However, whether and how PXR plays a role in colon cancer apoptosis has not been reported. In this study, we showed that activation of PXR by genetic (using a constitutively activated PXR) or pharmacological (using PXR agonist rifampicin) means protected the PXR-overexpressing colon cancer HCT116 cells from deoxycholic acid-induced apoptosis. Interestingly, activation of PXR also protected HCT116 cells from adriamycin-induced cell death, suggesting that the antiapoptotic effect of PXR was not bile acid specific. Moreover, the antiapoptotic effect of PXR in HCT116 cells appeared to be independent of xenobiotic enzyme regulation, because these cells had little basal and inducible expression of bile acid-detoxifying enzymes. Instead, SuperArray analysis showed that PXR-mediated deoxycholic acid resistance was associated with up-regulation of multiple antiapoptotic genes, including BAG3, BIRC2, and MCL-1, and down-regulation of proapoptotic genes, such as BAK1 and TP53/p53. Treatment with rifampicin in colon cancer LS180 cells, a cell line known to express endogenous PXR, also inhibited apoptosis. Activation of PXR in transgenic mice inhibited bile acid-induced colonic epithelial apoptosis and sensitized mice to dimethylhydrazine-induced colonic carcinogenesis, suggesting that the antiapoptotic effect of PXR is conserved in normal colon epithelium. In summary, our results have established the antiapoptotic role of PXR in both human colon cancer cells and normal mouse colon epithelium.     

E2F1-directed activation of Bcl-2 is correlated with lactoferrin-induced apoptosis in Jurkat leukemia T lymphocytes

Lactoferrin (Lf) has been shown to control the proliferation of a variety of mammalian cells. Recently, we reported that human Lf induces apoptosis via a c-Jun N-terminal kinases (JNK)-associated Bcl-2 pathway that stimulates programmed cell death. In order to gain insight into the mechanism underlying Lf-triggered apoptotic features, we attempted to determine the mechanisms whereby the Lf-induced Bcl-2 family proteins exert their pro- or anti-apoptotic effects in Jurkat leukemia T lymphocytes. Treatment of the cells with high concentrations of Lf resulted in a significant reduction in in vitro growth and cell viability. As the levels of Lf increased, greater quantities of CDK6 and hyper-phosphorylated retinoblastoma protein were produced, resulting in the induction of E2F1-dependent apoptosis. Simultaneously, PARP and caspases were efficiently cleaved during Lf-induced apoptosis. The E2F1-induced apoptotic process occurred preferentially in p53-deficient Jurkat leukemia cells. Therefore, we attempted to determine whether E2F1-regulated Bcl-2 family proteins involved in the apoptotic process were relevant to Lf-induced apoptosis. We found that Lf increased the interaction of Bcl-2 with the pro-apoptotic protein Bad, whereas the total protein levels did not change significantly. Our results, collectively, suggest that Lf exploits the control mechanism of E2F1-regulated target genes or Bcl-2 family gene networks involved in the apoptotic process in Jurkat human leukemia T lymphocytes.     

Synthesis and biological evaluation of a new class of glycoconjugated disulfides that exhibit potential anticancer properties

A synthetic strategy, based on the in situ generation of sulfenic acids and their thermolysis in the presence of thiols, was developed for obtaining a collection of polyvalent disulfides in which a benzene scaffold accommodates two or three flexible arms connecting saccharide moieties. Targeting carbohydrate metabolism or carbohydrate-binding proteins may constitute important approaches in the discovery process of new therapeutic anticancer agents. Therefore, a preliminary screening to ascertain the cytostatic/cytotoxic potential of this new class of enantiopure glycoconjugated disulfides has been conducted. Among them, products with two disulfide arms, harbouring galactose rings, induced high levels of apoptosis on U937 histiocytic lymphoma cells, but lower levels of cell death on peripheral blood mononuclear cells from healthy donors. Further experiments indicated that apoptosis induced by these glycoconjugated bis(disulfides) in U937 cells corresponds to the Bcl-2-sensitive, intrinsic form of apoptotic cell death. The bioinvestigation was extended to a panel of human cancer cell lines with different levels of malignancy and resistance to chemotherapeutic agents. Compounds under study proved to induce detectable levels of cell death towards all the tested cancer cell lines.     

The Na+/H+ exchanger controls deoxycholic acid-induced apoptosis by a H+-activated, Na+-dependent ionic shift in esophageal cells

Apoptosis resistance is a hallmark of cancer cells. Typically, bile acids induce apoptosis. However during gastrointestinal (GI) tumorigenesis the cancer cells develop resistance to bile acid-induced cell death. To understand how bile acids induce apoptosis resistance we first need to identify the molecular pathways that initiate apoptosis in response to bile acid exposure. In this study we examined the mechanism of deoxycholic acid (DCA)-induced apoptosis, specifically the role of Na(+)/H(+) exchanger (NHE) and Na(+) influx in esophageal cells. In vitro studies revealed that the exposure of esophageal cells (JH-EsoAd1, CP-A) to DCA (0.2 mM-0.5 mM) caused lysosomal membrane perturbation and transient cytoplasmic acidification. Fluorescence microscopy in conjunction with atomic absorption spectrophotometry demonstrated that this effect on lysosomes correlated with influx of Na(+), subsequent loss of intracellular K(+), an increase of Ca(2+) and apoptosis. However, ethylisopropyl-amiloride (EIPA), a selective inhibitor of NHE, prevented Na(+), K(+) and Ca(2+) changes and caspase 3/7 activation induced by DCA. Ouabain and amphotericin B, two drugs that increase intracellular Na(+) levels, induced similar changes as DCA (ion imbalance, caspase3/7 activation). On the contrary, DCA-induced cell death was inhibited by medium with low a Na(+) concentrations. In the same experiments, we exposed rat ileum ex-vivo to DCA with or without EIPA. Severe tissue damage and caspase-3 activation was observed after DCA treatment, but EIPA almost fully prevented this response. In summary, NHE-mediated Na(+) influx is a critical step leading to DCA-induced apoptosis. Cells tolerate acidification but evade DCA-induced apoptosis if NHE is inhibited. Our data suggests that suppression of NHE by endogenous or exogenous inhibitors may lead to apoptosis resistance during GI tumorigenesis.     

Deoxycholate induces DNA damage and apoptosis in human colon epithelial cells expressing either mutant or wild-type p53

Diets rich in fat result in higher concentrations of secondary bile acids or their salts in the colon, which may adversely affect cells of the colonic epithelium. Because secondary bile acids are thought to be genotoxic, exposing colon epithelial cells to secondary bile acids may induce DNA damage that might lead to apoptosis. The requirement for the p53 tumor suppressor gene in such events is unknown. In particular, the effects of secondary bile acids on colon epithelial cells having different p53 tumor suppressor gene status have not been examined. Therefore, HCT-116 and HCT-15 human colon adenocarcinoma cells, which express the wild-type and mutant p53 genes, respectively, were exposed to physiological concentrations of deoxycholate. The cells were then analyzed for evidence of DNA damage and apoptosis. After 2 h of incubation with 300 microM deoxycholate, both cell lines had greater levels of single-strand breaks in DNA as assessed by the comet assay. After 6 h of exposure to deoxycholate, HCT-116 and HCT-15 cells showed morphological signs of apoptosis, i.e., membrane blebbing and the presence of apoptotic bodies. Chromatin condensation and fragmentation were also seen after staining DNA with 4',6-diamidino-2-phenylindole. Other apoptotic assays revealed greater binding of annexin V-fluorescein isothiocyanate, as well as greater post-enzymatic labeling with dUTP-fluorescein isothiocyanate, by both cell lines exposed to deoxycholate. These data suggest that deoxycholate caused DNA damage in colon epithelial cells that was sufficient to trigger apoptosis in a p53-independent manner.     

Antisense inhibition of BCL-2 expression induces retinoic acid-mediated cell death during differentiation of human NT2N neurons

Changes in expression of the proto-oncogene Bcl-2 are well known in the developing brain, with a high expression level in young post-mitotic neurons that are beginning the outgrowth of processes. The physiological significance of the Bcl-2 up-regulation in these neurons is not fully understood. We used a differentiation model for human CNS neurons to study the expression and function of Bcl-2. NT2/D1 human neuronal precursor cells differentiated into a neuronal phenotype in the presence of 10 microM retinoic acid for 3-5 weeks. This concentration of retinoic acid was not toxic to undifferentiated NT2/D1 cells but was sufficient to up-regulate the BCL-2 protein in 6 days. The BCL-2 levels increased further after 3 weeks, i.e. when the cells started to show neuronal morphology. Inhibition of the accumulation of endogenous BCL-2 with vectors expressing the antisense mRNA of Bcl-2 caused extensive apoptosis after 3 weeks of the retinoic acid treatment. The loss of neuron-like cells from differentiating cultures indicated that the dead cells were those committed to neuronal differentiation. Death was related to the presence of retinoic acid since withdrawal of retinoic acid after 16 days of treatment dramatically increased cell surviving. The ability of BCL-2 to prevent retinoic acid-induced cell death was also confirmed in undifferentiated NT2/D1 cells that were transfected with a vector containing Bcl-2 cDNA in sense orientation and exposed to toxic doses (40-80 microM) of retinoic acid. Furthermore, down-regulation of BCL-2 levels by an antisense oligonucleotide in neuronally differentiated NT2/D1 cells increased their susceptibility to retinoic acid-induced apoptosis. These results indicate that one function of the up-regulation of endogenous BCL-2 during neuronal differentiation is to regulate the sensitivity of young post-mitotic neurons to retinoic acid-mediated apoptosis.     

SAHA treatment overcomes the anti-apoptotic effects of Bcl-2 and is associated with the formation of mature PML nuclear bodies in human leukemic U937 cells

Bcl-2 protects tumor cells from the apoptotic effects of various antineoplastic agents. Increased expression of Bcl-2 has been associated with poor response to chemotherapy in various malignancies, including leukemia. Therefore, bypassing the resistance conferred by anti-apoptotic factors such as Bcl-2 represents an attractive therapeutic strategy against cancer cells, including leukemic cells. We undertook this study to examine whether SAHA (suberoylanilide hydroxamic acid) overcomes the resistance by Bcl-2 in human leukemic cells, with a specific focus on the involvement of PML-NBs. Experiments were conducted with Bcl-2-overexpressing human leukemic U937 cells. Since we previously demonstrated that overexpression of Bcl-2 attenuates resveratrol-induced apoptosis in human leukemic U937 cells, resveratrol-treated U937 cells were used as a negative control. The present study indicates that SAHA at 1-7 μM, the dose range known to induce apoptosis in various cancer cells, overcomes the anti-apoptotic effects of Bcl-2 in Bcl-2-overexpressing human leukemic U937 cells. Notably, we observed that SAHA-induced formation of mature promyelocytic leukemia (PML) nuclear bodies (NBs) correlates with overcoming the anti-apoptotic effects of Bcl-2 in human leukemic U937 cells. Thus, PML protein and the formation of mature PML-NBs could be considered as therapeutic targets that could help bypass the resistance to apoptosis conferred by Bcl-2. Elucidating exactly how PML regulates Bcl-2 will require further work.     

Mechanisms of drug sensitization to TRA-8, an agonistic death receptor 5 antibody, involve modulation of the intrinsic apoptotic pathway in human breast cancer cells

TRA-8, a monoclonal antibody to death receptor 5 induces apoptosis in various cancer cells; however, the degree of sensitivity varies from highly sensitive to resistant. We have previously shown that resistance to TRA-8 can be reversed by using chemotherapeutic agents, but the mechanism underlying this sensitization was not fully understood. Here, we examined the combination of TRA-8 with doxorubicin or bortezomib in breast cancer cells. In TRA-8-resistant BT-474 and T47D cells, both chemotherapy agents synergistically sensitized cells to TRA-8 cytotoxicity with enhanced activation of apoptosis shown by cleavage of caspases and PARP, reduced Bid, increased proapoptotic Bcl-2 proteins, and increased mitochondrial membrane depolarization. Doxorubicin or bortezomib combined with TRA-8 also reduced Bcl-XL and X-linked inhibitors of apoptosis (XIAP) in treated cells. Furthermore, targeting these proteins with pharmacologic modulators, AT-101, BH3I-2' and AT-406, produced sensitization to TRA-8. TRA-8 combined with AT-101 or BH3I-2', inhibitors of antiapoptotic Bcl-2 proteins, produced synergistic cytotoxicity against ZR-75-1, BT-474, and T47D cells. The IAP-targeting compound, AT-406, was synergistic with TRA-8 in BT-474 cells, and to a lesser extent T47D cells. Activation of the intrinsic apoptotic pathway was a common mechanism associated with sensitization of TRA-8-resistant breast cancer cell lines. Collectively, these studies show that the Bcl-2 and IAP families of proteins are involved in TRA-8 and chemotherapy resistance via their modulation of the intrinsic apoptotic pathway. Targeting these proteins with novel agents sensitized TRA-8-resistant breast cancer cells, suggesting this approach may represent a potent therapeutic strategy in the treatment of breast cancer.     

The cellular concentration of Bcl-2 determines its pro- or anti-apoptotic effect

Bcl-2 is an oncoprotein that is widely known to promote cell survival by inhibiting apoptosis. We explored the consequences of different expression paradigms on the cellular action of Bcl-2. Using either transient or stable transfection combined with doxycycline-inducible expression, we titrated the cellular concentration of Bcl-2. With each expression paradigm Bcl-2 was correctly targeted to the endoplasmic reticulum and mitochondria. However, with protocols that generated the greatest cellular concentrations of Bcl-2 the structure of these organelles was dramatically altered. The endoplasmic reticulum appeared to be substantially fragmented, whilst mitochondria coalesced into dense perinuclear structures. Under these conditions of high Bcl-2 expression, cells were not protected from pro-apoptotic stimuli. Rather Bcl-2 itself caused a significant amount of spontaneous cell death, and sensitised the cells to apoptotic agents such as staurosporine or ceramide. We observed a direct correlation between Bcl-2 concentration and spontaneous apoptosis. Expression of calbindin, a calcium buffering protein, or an enzyme that inhibited inositol 1,4,5-trisphosphate-mediated calcium release, significantly reduced cell death caused by Bcl-2 expression. We further observed that high levels of Bcl-2 expression caused lipid peroxidation and that the deleterious effects of Bcl-2 could be abrogated by the reactive oxygen species (ROS) scavenger Trolox. When stably expressed at low levels, Bcl-2 did not corrupt organelle structure or trigger spontaneous apoptosis. Rather, it protected cells from pro-apoptotic stimuli. These data reveal that high cellular concentrations of Bcl-2 lead to a calcium- and ROS-dependent induction of death. Selection of the appropriate expression paradigm is therefore crucial when investigating the biological role of Bcl-2.     

2,3,7,8-Tetrachlorodibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxin regulates Bovine Herpesvirus type 1 induced apoptosis by modulating Bcl-2 family members

Exposure to environmental contaminants, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), leads to an increased susceptibility to infectious agents. Infection of bovine cells (MDBK) with Bovine Herpesvirus 1 (BHV-1) anticipates virus-induced apoptosis, suggesting an involvement of TCDD in virus infection. Herein we analyzed the effects of TCDD on apoptotic pathway in MDBK cells infected with BHV-1. After 12 h of infection, TCDD induced a significant increase in apoptotic cells. TCDD caused a dose-dependent up-regulation and anticipated activation of caspases 3, 8 and 9, with respect to unexposed groups. TCDD anticipated cleavage of PARP, compared to controls. Furthermore TCDD increased Bax and Bid levels, and decreased Bcl-2 and Bcl-XL levels. Such events took place earlier in exposed than unexposed cells. These results showed that TCDD influences BHV-1 induced apoptosis through members of Bcl-2 family and up-regulating activation of caspases.     

Stiffness changes of tumor HEp2 cells correlates with the inhibition and release of TRAIL-induced apoptosis pathways

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) is a promising apoptotic agent that can selectively act on tumor cells. However, some cancer cells are resistant to TRAIL mediated apoptosis. In specific type of cells, sensitization by chemotherapeutic drugs may overcome the resistance to TRAIL induced apoptosis. In this work, atomic force microscopy (AFM) nanoindentation spectroscopy combined with fluorescence methods were used to investigate the biomechanical aspects of the resistance and unblocking of apoptosis in larynx carcinoma HEp2 cells treated with TRAIL. It is shown that there is a direct correlation between the increase in mechanical cell stiffness and the inhibition of apoptosis induced by TRAIL in HEp2 cells. Conversely, unblocking of apoptosis by sensitization of HEp2 cells with a chemotherapeutic drug Actinomycin D is related to the depolymerization of F-actin and to the decrease in the cell stiffness. Both effects, that is, changes in the mechanical stiffness of the cell and the inhibition of apoptotic pathway, are closely related to the Bcl-2 activity. Most probably, the depolymerization of F-actin results from downregulation of Rho protein, which in turn is accompanied by a lower activity of Bcl-2 and in consequence releases the intrinsic apoptotic channel. The presented results reveal a promising application of nanoindentation spectroscopy with an AFM tip as a novel tool for monitoring the processes of apoptosis inhibition.     

Dual anticancer activity of 8-Cl-cAMP: inhibition of cell proliferation and induction of apoptotic cell death

8-Cl-cAMP induces apoptotic cell death in human cancer cells. To look at this more closely, we examined the changes in the levels of Bcl-2 family proteins during 8-Cl-cAMP-induced apoptosis of SH-SY5Y human neuroblastoma cells. Following the treatment with 8-Cl-cAMP, Bcl-2 was transiently down-regulated and Bad was increased continuously up to day 5. In addition, overexpression of Bcl-2 efficiently blocked the 8-Cl-cAMP-induced apoptosis, suggesting Bcl-2 family proteins may be involved in the 8-Cl-cAMP-induced apoptosis. The contribution of the apoptotic cell death and the inhibition of cell proliferation in the 8-Cl-cAMP-induced growth inhibition was closely monitored in the Bcl-2-overexpressing cells. Though the apoptosis was reduced significantly, no significant difference was observed in the inhibition of cell proliferation up to day 2 of 8-Cl-cAMP treatment. These results suggest that 8-Cl-cAMP exerts anticancer activity by two distinct mechanisms, i.e. , through the inhibition of cell proliferation as well as the induction of apoptosis. Supporting this notion was the observations that (1) suppression of apoptosis by zVAD did not abrogate 8-Cl-cAMP-induced inhibition of cell proliferation, and (2) 8-Cl-cAMP did not show additive inhibition of cell proliferation in RIIbeta-overexpressing cells.