Inhibition of autophagy enhances the anticancer activity of silver nanoparticles

Silver nanoparticles (Ag NPs) are cytotoxic to cancer cells and possess excellent potential as an antitumor agent. A variety of nanoparticles have been shown to induce autophagy, a critical cellular degradation process, and the elevated autophagy in most of these situations promotes cell death. Whether Ag NPs can induce autophagy and how it might affect the anticancer activity of Ag NPs has not been reported. Here we show that Ag NPs induced autophagy in cancer cells by activating the PtdIns3K signaling pathway. The autophagy induced by Ag NPs was characterized by enhanced autophagosome formation, normal cargo degradation, and no disruption of lysosomal function. Consistent with these properties, the autophagy induced by Ag NPs promoted cell survival, as inhibition of autophagy by either chemical inhibitors or ATG5 siRNA enhanced Ag NPs-elicited cancer cell killing. We further demonstrated that wortmannin, a widely used inhibitor of autophagy, significantly enhanced the antitumor effect of Ag NPs in the B16 mouse melanoma cell model. Our results revealed a novel biological activity of Ag NPs in inducing cytoprotective autophagy, and inhibition of autophagy may be a useful strategy for improving the efficacy of Ag NPs in anticancer therapy.     

Ibuprofen enhances the anticancer activity of cisplatin in lung cancer cells by inhibiting the heat shock protein 70

Hsp70 is often overexpressed in cancer cells, and the selective cellular survival advantage that it confers may contribute to the process of tumour formation. Thus, the pharmacological manipulation of Hsp70 levels in cancer cells may be an effective means of preventing the progression of tumours. We found that the downregulation of Hsp70 by ibuprofen in vitro enhances the antitumoural activity of cisplatin in lung cancer. Ibuprofen prominently suppressed the expression of Hsp70 in A549 cells derived from lung adenocarcinoma and sensitized them to cisplatin in association with an increase in the mitochondrial apoptotic cascade, whereas ibuprofen alone did not induce cell death. The cisplatin-dependent events occurring up- and downstream of mitochondrial disruption were accelerated by treatment with ibuprofen. The increase in cisplatin-induced apoptosis caused by the depletion of Hsp70 by RNA interference is evidence that the increased apoptosis by ibuprofen is mediated by its effect on Hsp70. Our observations indicate that the suppression of Hsp70 by ibuprofen mediates the sensitivity to cisplatin by enhancing apoptosis at several stages of the mitochondrial cascade. Ibuprofen, therefore, is a potential therapeutic agent that might allow lowering the doses of cisplatin and limiting the many challenge associated with its toxicity and development of drug resistance.     

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.     

Strong electronic charge as an important factor for anticancer activity of chitooligosaccharides (COS)

Even though several studies report the importance of chitosan derivatives for their anticancer activity, no clear information is available to describe the relationship between their charge properties and observed activities. In this research, differently charged chitooligosaccharide (COS) derivatives were synthesized and their anticancer activities were studied using three cancer cell lines, HeLa, Hep3B and SW480. Neutral red and MTT cell viability studies revealed that, highly charged COS derivatives could significantly reduce cancer cell viability, regardless to the positive or negative charge. Further, fluorescence microscopic observations and DNA fragmentation studies confirmed that the anticancer effect of these highly charged COS derivatives were due to necrosis. However, the exact molecular mechanism for anticancer activity of strongly charged COS compared to their poorly charged counterparts is not clear.     

Chk1 inhibitor SCH 900776 enhances the antitumor activity of MLN4924 on pancreatic cancer

MLN4924 inhibits the cullin-RING ligases mediated ubiquitin-proteasome system, and has showed antitumor activities in preclinical studies, but its effects and mechanisms on pancreatic cancer (PC) remains elusive. We found that MLN4924 inhibited the proliferation and clonogenicity of PC cells, caused DNA damage, particularly double-strand breaks, and leaded to Chk1 activation and cell-cycle arrest. Chk1 inhibitor SCH 900776 alone exhibited minimal cytotoxicity, and caused no DNA damage on PC cells. But in the combination therapy, SCH 900776 enhanced the cytotoxicity and DNA damage caused by MLN4924, likely by abrogating G2/M arrest and promoting DNA re-replication. In vivo study on a xenograft PC mouse model also showed that SCH 900776 increased the efficacy of MLN4924. We also evaluated the level of NEDD8-activating enzyme (NAE), the direct target of MLN4924, and found that NAE level was elevated in PC tissues compared with normal pancreas, but was irrelevant with prognosis. Our findings provide the preclinical evidence and the rationale of the combination therapy of MLN4924 with SCH 900776 or other Chk1 inhibitors to treat PC.     

Thio-functionalized carbohydrate thiosemicarbazones and evaluation of their anticancer activity

Thiosemicarbazides and their analogs have shown potential medical applications as antiviral, antibacterial and anticancer drugs. We designed, synthesized and evaluated in vitro anticancer activity against ovarian (A2780), cervix (HeLa), colon (LoVo), breast (MCF-7) and brain (MO59J) human cancer cell lines of seven novel compounds –S-glycosylated thiosemicarbazones. We assessed the cyto- and genotoxic properties of all novel compounds using a variety of methods including comet assay, XTT assay, various fluorescent assays and toxicology PathwayFinder expression array. We tried to evaluate their possible mechanism of action with particular attention to induction of DNA damage and repair, apoptosis, oxidative stress analysis and cellular response in terms of changes in gene expression. The most sensitive cell line was human ovarian cancer. The results revealed that the major activity against A2780 cancer cell line displayed by our compounds is induction of DNA damage. This effect is not associated with apoptosis or oxidative stress induction and the resulting damage will not lead to cell cycle arrest. We also observed up-expression of heat shock related genes and NQO1 gene in response to our compounds. The second effect seems to be specific to glycosylated S-bond compounds as we observed it earlier. Upregulation of heat shock protein encoding genes suggest that our compounds induce stressful conditions. The nature of this phenomena (heat shock, pH shift or hypoxia) needs further study.     

Suicidal differential housekeeping gene activity in apoptosis induced by DCNP

Previous suggestions of CpG-specific apoptotic commitment implied critical epigenetic modulation of house-keeping genes which have canonical CpG islands at 5 promoter regions. Differential housekeeping gene activity however has not been shown. Using a focussed microarray (genechip) of 22 housekeeping genes we show this in apoptosis induced in human Chang liver cells by DCNP (2,6-dichloro-4-nitrophenol), a non-genotoxic inhibitor of sulfate detoxification. 3–7 folds downregulation of 9 genes in glycolysis, tricarboxylic acid cycle and the respiratory electron transport chain suggested gene-directed energy depletion which was correlated with observed ATP depletion. 4 folds downregulation of the pyruvate dehydrogenease gene suggested gene-directed metabolic acidosis which was correlated with observed cell acidification. Other differential housekeeping gene activity, including 4 folds upregulation of microtubular alpha-tubulin gene, and 2 folds upregulation of ubiquitin, also had a bearing on apoptosis. Broadspectrum zVAD-fmk caspase inhibition abolished 200 bp DNA ladder fragmentations but not the CpG-specific megabase fragmentations and other hallmarks of cell destruction, suggesting a caspase-independent cell death. Death appeared committed at gene-level.     

Critical role of poly(ADP‐ribose) polymerase‐1 in modulating the mode of cell death caused by continuous oxidative stress

Continuously generated hydrogen peroxide (H₂O₂) inhibits typical apoptosis and instead initiates a caspase‐independent, apoptosis‐inducing factor (AIF)‐mediated pyknotic cell death. This may be related to H₂O₂‐mediated DNA damage and subsequent ATP depletion, although the exact mechanisms by which the mode of cell death is decided after H₂O₂ exposure are still unclear. Accumulated evidence and our previous data led us to hypothesize that continuously generated H₂O₂, not an H₂O₂ bolus, induces severe DNA damage, signaling poly(ADP‐ribose) polymerase‐1 (PARP‐1) activation, ATP depletion, and eventually caspase‐independent cell death. Results from the present study support that H₂O₂ generated continuously by glucose oxidase causes excessive DNA damage and PARP‐1 activation. Blockage of PARP‐1 by a siRNA transfection or by pharmacological inhibitor resulted in the significant inhibition of ATP depletion, loss of mitochondrial membrane potential, nuclear translocation of AIF and endonuclease G, and eventually conversion to caspase‐dependent apoptosis. Overall, the current study demonstrates the different roles of PARP‐1 inhibition in modulation of cell death according to the method of H₂O₂ exposure, that is, continuous generation versus a direct addition. J. Cell. Biochem. 108: 989–997, 2009. © 2009 Wiley‐Liss, Inc.     

Urokinase-targeted recombinant bacterial protein toxins-a rationally designed and engineered anticancer agent for cancer therapy

Urokinase-targeted recombinant bacterial protein toxins are a sort of rationally designed and engineered anticancer recombinant fusion proteins representing a novel class of agents for cancer therapy.Bacterial protein toxins have long been known as the primary virulence factor(s) for a variety of pathogenic bacteria and are the most powerful human poisons.On the other hand,it has been well documented that urokinase-type plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR),making up the uPA system,are overexpressed in a variety of human tumors and tumor cell lines.The expression of uPA system is highly correlated with tumor invasion and metastasis.To exploit these characteristics in the design of tumor cell-selective cytotoxins,two prominent bacterial protein toxins,i.e.,the diphtheria toxin and anthrax toxin are deliberately engineered through placing a sequence targeted specifically by the uPA system to form anticancer recombinant fusion proteins.These uPA system-targeted bacterial protein toxins are activated selectively on the surface of uPA systemexpressing tumor cells,thereby killing these cells.This article provides a review on the latest progress in the exploitation of these recombinant fusion proteins as potent tumoricidal agents.It is perceptible that the strategies for cancer therapy are being innovated by this novel therapeutic approach.     

Characterization of cell clones stably transfected with short form caspase-9: Apoptotic resistance and Bcl-XL expression

Caspases play important roles in the initiation and progression of apoptosis. In experimental models of ATP depletion, we have demonstrated the activation of caspase-9, -8, and -3, which is followed by the development of apoptotic morphology. To determine the specific contribution of caspase-9 to ATP depletion-induced apoptosis, we transfected renal epithelial cells with its endogenous dominant-negative inhibitor caspase-9S. Two cell clones with stable transfection were obtained. These clones expressed caspase-9S, and the cytosol isolated from these cells was resistant to cytochrome c-induced caspase activation in vitro. The clones were then examined for ATP depletion-induced apoptosis. Compared with the wild-type cells, the caspase-9S clones were markedly resistant to apoptosis in this model. Caspase activation was also inhibited. Surprisingly, these clones also showed significantly less cytochrome c release during ATP-depletion. Moreover, Bax translocation to mitochondria was inhibited, suggesting that these clones were resistant to apoptosis not only at the cytosolic caspase activation level but also at the upstream mitochondrial level. To gain insights into the mitochondrial resistance, we analyzed the expression of Bcl-2 family proteins. While the expression of Bax, Bak, and Bcl-2 was comparable to the wild-type cells, the selected clones showed specific up-regulation of Bcl-XL, an anti-apoptotic protein. We conclude that the selected clones were resistant to apoptosis at two levels. In the cytosol, they expressed dominant negative caspase-9, and at the mitochondria they up-regulated Bcl-XL.     

KUD773, a phenylthiazole derivative,displays anticancer activity in human hormone-refractory prostate cancers through inhibition of tubulin polymerization and anti-Aurora A activity

Background

Hormone-refractory prostate cancer (HRPC), which is resistant to hormone therapy, is a major obstacle in clinical treatment. An approach to inhibit HRPC growth and ultimately to kill cancers is highly demanded.

Results

KUD773 induced the anti-proliferative effect and subsequent apoptosis in PC-3 and DU-145 (two HRPC cell lines); whereas, it showed less active in normal prostate cells. Further examination showed that KUD773 inhibited tubulin polymerization and induced an increase of mitotic phosphoproteins and polo-like kinase 1 (PLK1) phosphorylation, indicating a mitotic arrest of the cell cycle through an anti-tubulin action. The kinase assay demonstrated that KUD773 inhibited Aurora A activity. KUD773 induced an increase of Cdk1 phosphorylation at Thr¹⁶¹ (a stimulatory phosphorylation site) and a decrease of phosphorylation at Tyr¹⁵ (an inhibitory phosphorylation site), suggesting the activation of Cdk1. The data were substantiated by an up-regulation of cyclin B1 (a Cdk1 partner). Furthermore, KUD773 induced the phosphorylation and subsequent down-regulation of Bcl-2 and activation of caspase cascades.

Conclusions

The data suggest that KUD773 induces apoptotic signaling in a sequential manner. It inhibits tubulin polymerization associated with an anti-Aurora A activity, leading to Cdk1 activation and mitotic arrest of the cell cycle that in turn induces Bcl-2 degradation and a subsequent caspase activation in HRPCs.     

In vitro anticancer activity of loquat tea by inducing apoptosis in human leukemia cells

Fresh loquat leaves have been used as folk health herb in Asian countries for long time, although the evidence supporting their functions is still minimal. This study aimed to clarify the chemopreventive effect of loquat tea extract (LTE) by investigating the inhibition on proliferation, and underlying mechanisms in human promyelocytic leukemia cells (HL-60). LTE inhibited proliferation of HL-60 in a dose-dependent manner. Molecular data showed that the isolated fraction of LTE induced apoptosis of HL-60 as characterized by DNA fragmentation; activation of caspase-3, -8, and -9; and inactivation of poly(ADP)ribose polymerase. Moreover, LTE fraction increased the ratio of pro-apoptotic Bcl-2-associated X protein (Bax)/anti-apoptotic myeloid cell leukemia 1 (Mcl-1) that caused mitochondrial membrane potential loss and cytochrome c released to cytosol. Thus, our data indicate that LTE might induce apoptosis in HL-60 cells through a mitochondrial dysfunction pathway. These findings enhance our understanding for chemopreventive function of loquat tea.     

Vitamin E analogs: A new class of multiple action agents with anti-neoplastic and anti-atherogenic activity

The incidence of cancer and atherosclerosis, two most common causes of death in developed countries, has been stagnating or, even, increasing. Drugs effective against such conditions are needed and, in this regard, the potential anti-atherosclerotic activity of vitamin E analogs has been studied extensively. Surprisingly, recent results indicate that these agents may also exert anti-neoplastic effects. Here we review the evidence that particular analogs of vitamin E may act as both antiatherogenic and anti-cancer agents, and discuss the possible molecular bases for these actions.     

Denbinobin-mediated anticancer effect in human K562 leukemia cells: role in tubulin polymerization and Bcr-Abl activity

Denbinobin (5-hydroxy-3,7-dimethoxy-1,4-phenanthraquinone) has been reported to exhibit anti-tumor and anti-inflammatory activity. Nevertheless, the anti-tumor mechanism of denbinobin remains unclear. In the present study, we evaluated the anticancer activity of denbinobin in human myelogenous K562 leukemia cells. In accordance with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, we demonstrated that denbinobin inhibited cell viability in a concentration-dependent manner with an IC₅₀ value of 1.84M. Cell cycle analysis illustrated that exposure of denbinobin caused a G2/M phase accumulation in a time-dependent manner. Tubulin polymerization in cells was apparently enhanced by denbinobin, implying that denbinobin might have a regulatory role in tubulin/microtubule. Furthermore, denbinobin significantly suppressed the expression of Bcr-Abl and phosphorylation of CrkL, a crucial tyrosine kinase and an adaptor protein in chronic myeloid leukemia, respectively. Denbinobin also markedly enhanced CD11b expression after a long-term treatment, suggesting that denbinobin might play a role in facilitating differentiation in K562 cells. In summary, we have demonstrated that denbinobin displays anticancer effects in K562 cells through the increase of levels of tubulin polymerization and deregulation of Bcr-Abl signaling. Our data demonstrate that denbinobin could be a potential anticancer lead compound for further development.     

Arachidonic acid converts the glutathione depletion-induced apoptosis to necrosis by promoting lipid peroxidation and reducing caspase-3 activity in rat glioma cells

Intracellular glutathione (GSH) depletion induced by buthionine sulfoximine (BSO) caused cell death that seemed to be apoptosis in C6 rat glioma cells. Arachidonic acid (AA) promoted BSO-induced cell death by accumulating reactive oxygen species (ROS) or hydroperoxides. AA inhibited caspase-3 activation and internucleosomal DNA fragmentation during the BSO-induced GSH depletion. Furthermore, AA reduced intracellular ATP content, induced dysfunction of mitochondrial membrane and enhanced 8-hydroxy-2'-deoxyguanosine (8-OH-dG) production. There was significant increase of 12-lipoxygenase activity in the presence of AA under the BSO-induced GSH depletion in C6 cells. These results suggest that AA promotes cell death by changing to necrosis from apoptosis through lipid peroxidation initiated by lipid hydroperoxides produced by 12-lipoxygenase under the GSH depletion in C6 cells. Some ROS such as hydroperoxide produced by unknown pathway make hydroxy radicals and induce 8-OH-dG formation in the cells. The conversion of apoptosis to necrosis may be a possible event under GSH depleted conditions.     

Inhibition of p38 mitogen-activated protein kinase reduces TNF-induced activation of NF-kappaB, elicits caspase activity, and enhances cytotoxicity

Among other cellular responses, tumor necrosis factor (TNF) induces different forms of cell death and the activation of the p38 mitogen-activated protein kinase (MAPK). The influence of p38 MAPK activation on TNF-induced apoptosis or necrosis is controversially discussed. Here, we demonstrate that pharmacological inhibition of p38 MAPK enhances TNF-induced cell death in murine fibroblast cell lines L929 and NIH3T3. Furthermore, overexpression of dominant-negative versions of p38 MAPK or its upstream kinase MKK6 led to increased cell death in L929 cells. While overexpression of the p38 isoforms alpha and beta did not protect L929 cells from TNF-induced toxicity, overexpression of constitutively active MKK6 decreased TNF-induced cell death. Although the used inhibitors of p38 MAPK decreased the phosphorylation of the survival kinase PKB/Akt, this effect could be ruled out as cause of the observed sensitization to TNF-induced cytotoxicity. Finally, we demonstrate that the nuclear factor kappaB (NF-kappaB)-dependent gene expression, shown as an example for the anti-apoptotic gene cellular inhibitor of apoptosis (c-IAP2), was reduced by p38 MAPK inhibition. In consequence, we found that inhibition of p38 MAPK led to the activation of the executioner caspase-3.     

Increased local vascular endothelial growth factor expression associated with antitumor activity of proteasome inhibitor

Inhibition of the proteasome, a multicatalytic proteinase complex, is an attractive approach to cancer therapy. Here we report that a selective inhibitor of the chymotrypsin-like activity of the proteasome, PSI (N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal) may inhibit growth of solid tumors not only through apoptosis induction, but also indirectly--through inhibition of angiogenesis. Two murine tumors: colon adenocarcinoma (C-26) and Lewis lung carcinoma (3LL) were chosen to study the antitumor effect of PSI. In an in vivo model of local tumor growth, PSI exerted significant antitumor effects against C-26 colon carcinoma, but not against 3LL lung carcinoma. Retardation of tumor growth was observed in mice treated with both 10 nmoles and 100 nmoles doses of PSI and in the latter group prolongation of the survival time of tumor-bearing mice was observed. PSI inhibited angiogenesis in the C-26 growing tumors with no such effect in 3LL tumors. Unexpectedly, that activity was associated with upregulation of vascular endothelial growth factor (VEGF) at the level of mRNA expression and protein production in C-26 tumors treated with PSI. C-26 cells treated with PSI produced increased amounts of VEGF in vitro in a dose- and time-dependent manner. We demonstrated that in C-26 colon adenocarcionoma higher VEGF production may render endothelial cells susceptible to the proapoptotic activity of PSI and is associated with inhibition of tumor growth.     

A novel monoterpenoid indole alkaloid with anticancer activity from Melodinus khasianus

A novel monoterpenoid indole alkaloid with unprecedented 6/5/6/4/6 fused rings, khasuanine A (1), was isolated from the roots of Melodinus khasianus. The structure was determined by extensive analysis of its HR-MS, 1D-, and 2D-NMR spectra. Khasuanine A markedly inhibited the proliferation of PC3 cell with IC₅₀ value of 0.45 μM. Further study showed that khasuanine A was able to induce the apoptosis of PC3 cells by activation of caspase 3 and p53, and by inhibition of Bcl-2.     

Plagiochin E,an antifungal bis(bibenzyl), exerts its antifungal activity through mitochondrial dysfunction-induced reactive oxygen species accumulation in Candida albicans

Background

Plagiochin E (PLE) is an antifungal macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L. Its antifungal mechanism is unknown. To elucidate the mechanism of action, its effect on mitochondria function in Candida albicans was studied.

Methods

We assayed the mitochondrial membrane potential (mtΔψ) using rhodamine 123, measured ATP level in mitochondria by HPLC, and detected the activities of mitochondrial F₀F₁-ATPase and dehydrogenases. Besides, the mitochondrial dysfunction-induced reactive oxygen species (ROS) production was determined by a fluorometric assay, and the effects of antioxidant L-cysteine on PLE-induced ROS production and the antifungal effect of PLE on C. albicans were also investigated.

Results

Exposure to PLE resulted in an elevation of mtΔψ, and a decrease of ATP level in mitochondria. The ATP depletion owed to PLE-induced enhancement of mitochondrial F₀F₁-ATPase and inhibition of the mitochondrial dehydrogenases. These dysfunctions of mitochondria caused ROS accumulation in C. albicans, and this increase in the level of ROS production and PLE-induced decrease in cell viability were prevented by addition of L-cysteine, indicating that ROS was an important mediator of the antifungal action of PLE.

Conclusions

PLE exerts its antifungal activity through mitochondrial dysfunction-induced ROS accumulation in C. albicans.

General significance

The effect of PLE on the mitochondria function in C. albicans was assayed for the first time. These results would conduce to elucidate its underlying antifungal mechanism.