Corilagin inhibits breast cancer growth via reactive oxygen species‐dependent apoptosis and autophagy

Corilagin is a component of Phyllanthus urinaria extract and has been found of possessing anti‐inflammatory, anti‐oxidative, and anti‐tumour properties in clinic treatments. However, the underlying mechanisms in anti‐cancer particularly of its induction of cell death in human breast cancer remain undefined. Our research found that corilagin‐induced apoptotic and autophagic cell death depending on reactive oxygen species (ROS) in human breast cancer cell, and it occurred in human breast cancer cell (MCF‐7) only comparing with normal cells. The expression of procaspase‐8, procaspase‐3, PARP, Bcl‐2 and procaspase‐9 was down‐regulated while caspase‐8, cleaved PARP, caspase‐9 and Bax were up‐regulated after corilagin treatment, indicating apoptosis mediated by extrinsic and mitochondrial pathways occurred in MCF‐7 cell. Meanwhile, autophagy mediated by suppressing Akt/mTOR/p70S6K pathway was detected with an increase in autophagic vacuoles and LC3‐II conversion. More significantly, inhibition of autophagy by chloroquine diphosphate salt (CQ) remarkably enhanced apoptosis, while the caspase inhibitor z‐VAD‐fmk failed in affecting autophagy, suggesting that corilagin‐induced autophagy functioned as a survival mechanism in MCF‐7 cells. In addition, corilagin induced intracellular reactive oxygen species (ROS) generation, when reduced by ROS scavenger NAC, apoptosis and autophagy were both down‐regulated. Nevertheless, in SK‐BR3 cell which expressed RIP3, necroptosis inhibitor Nec‐1 could not alleviate cell death induced by corilagin, indicating necroptosis was not triggered. Subcutaneous tumour growth in nude mice was attenuated by corilagin, consisting with the results in vitro. These results imply that corilagin inhibits cancer cell proliferation through inducing apoptosis and autophagy which regulated by ROS release.     

Carnosine Inhibits the Proliferation of Human Gastric Cancer SGC-7901 Cells through Both of the Mitochondrial Respiration and Glycolysis Pathways

Carnosine, a naturally occurring dipeptide, has been recently demonstrated to possess anti-tumor activity. However, its underlying mechanism is unclear. In this study, we investigated the effect and mechanism of carnosine on the cell viability and proliferation of the cultured human gastric cancer SGC-7901 cells. Carnosine treatment did not induce cell apoptosis or necrosis, but reduced the proliferative capacity of SGC-7901 cells. Seahorse analysis showed SGC-7901 cells cultured with pyruvate have active mitochondria, and depend on mitochondrial oxidative phosphorylation more than glycolysis pathway for generation of ATP. Carnosine markedly decreased the absolute value of mitochondrial ATP-linked respiration, and reduced the maximal oxygen consumption and spare respiratory capacity, which may reduce mitochondrial function correlated with proliferative potential. Simultaneously, carnosine also reduced the extracellular acidification rate and glycolysis of SGC-7901 cells. Our results suggested that carnosine is a potential regulator of energy metabolism of SGC-7901 cells both in the anaerobic and aerobic pathways, and provided a clue for preclinical and clinical evaluation of carnosine for gastric cancer therapy.     

Salvianolic acid B inhibits ototoxic drug–induced ototoxicity by suppression of the mitochondrial apoptosis pathway

It has been claimed that salvianolic acid B (Sal B), a natural bioactive antioxidant, exerts protective effects in various types of cells. This study aims to evaluate the antioxidant and anti‐apoptosis effects of Sal B in a cultured HEI‐OC1 cell line and in transgenic zebrafish (Brn3C: EGFP). A CCK‐8 assay, Annexin V Apoptosis Detection Kit, TUNEL and caspase‐3/7 staining, respectively, examined apoptosis and cell viability. The levels of reactive oxygen species (ROS) were evaluated by CellROX and MitoSOX Red staining. JC‐1 staining was employed to detect the mitochondrial membrane potential (ΔΨm). Western blotting was used to assess expressions of Bax and Bcl‐2. The expression pattern of p‐PI3K and p‐Akt was determined by immunofluorescent staining. We found that Sal B protected against neomycin‐ and cisplatin‐induced apoptotic features, enhanced cell viability and accompanied with decreased caspase‐3 activity in the HEI‐OC1 cells. Supplementary experiments determined that Sal B reduced ROS production (increased ΔΨm), promoted Bcl‐2 expression and down‐regulated the expression of Bax, as well as activated PI3K/AKT signalling pathways in neomycin‐ and cisplatin‐injured HEI‐OC1 cells. Moreover, Sal B markedly decreased the TUNEL signal and protected against neomycin‐ and cisplatin‐induced neuromast HC loss in the transgenic zebrafish. These results unravel a novel role for Sal B as an otoprotective agent against ototoxic drug–induced HC apoptosis, offering a potential use in the treatment of hearing loss.     

ω‐hydroxyundec‐9‐enoic acid induces apoptosis by ROS mediated JNK and p38 phosphorylation in breast cancer cell lines

ω‐Hydroxyundec‐9‐enoic acid (ω‐HUA), a plant secondary metabolite, exhibits anti‐fungal activity. However, its effect on breast cancer cells is unknown. Here, we investigated the anti‐ breast cancer activity of ω‐HUA and its underlying mechanism. Treatment of human breast cancer cell lines, MDA‐MB‐231 and MDA‐MB‐435, with ω‐HUA induced apoptotic cell death with increased cleaved caspase‐3 and poly (ADP‐ribose) polymerase (PARP) levels, and p38 and JNK phosphorylation. Inhibition of these mitogen‐activated protein kinase (MAPK) pathways using specific inhibitors or siRNA, for p38 and JNK, respectively, blocked the ω‐HUA‐induced apoptosis in a dose‐dependent manner. Moreover, pretreatment of the cells with antioxidant N‐acetyl cysteine (NAC) inhibited ω‐HUA‐induced increased reactive oxygen species (ROS) levels, cleaved caspase‐3 and cleaved PARP, and phosphorylated JNK, phosphorylated p38, and increased cell viability and colony‐forming ability. MDA‐MB‐231 xenograft model showed that the ω‐HUA‐treated group exhibited greater tumor regression and significantly reduced tumor weight compared to that exhibited by the vehicle‐administered group. Collectively, ω‐HUA‐induced intracellular ROS generation induced breast cancer cell apoptosis through JNK and p38 signaling pathway activation, resulting in tumor regression. The results suggested that ω‐HUA is an effective supplement for inhibiting human breast cancer growth.     

Carnosine protects against permanent cerebral ischemia in histidine decarboxylase knockout mice by reducing glutamate excitotoxicity

Recently, we showed that carnosine protects against NMDA-induced excitotoxicity in differentiated PC12 cells through a histaminergic pathway. However, whether the protective effect of the carnosine metabolic pathway also occurs in ischemic brain is unknown. Utilizing the model of permanent middle cerebral artery occlusion (pMCAO) in mice, we found that carnosine significantly improved neurological function and decreased infarct size in both histidine decarboxylase knockout and the corresponding wild-type mice to the same extent. Carnosine decreased the glutamate levels and preserved the expression of glutamate transporter-1 (GLT-1) but not the glutamate/aspartate transporter in astrocytes exposed to ischemia in vivo and in vitro. It suppressed the dissipation of ΔΨ_m and generation of mitochondrial reactive oxygen species (ROS) induced by oxygen–glucose deprivation in astrocytes. Furthermore, carnosine also decreased the mitochondrial ROS and reversed the decrease in GLT-1 induced by rotenone. These findings are the first to demonstrate that the mechanism of carnosine action in pMCAO may not be mediated by the histaminergic pathway, but by reducing glutamate excitotoxicity through the effective regulation of the expression of GLT-1 in astrocytes due to improved mitochondrial function. Thus, our study reveals a novel antiexcitotoxic agent in ischemic injury.     

Anticancer activities of genistein‐topotecan combination in prostate cancer cells

Prostate cancer is one of the leading causes of death in men aged 40 to 55. Genistein isoflavone (4′, 5′, 7‐trihydroxyisoflavone) is a dietary phytochemical with demonstrated anti‐tumour activities in a variety of cancers. Topotecan Hydrochloride (Hycamtin) is an FDA‐approved chemotherapy drug, primarily used for secondary treatment of ovarian, cervical and small cell lung cancers. This study was to demonstrate the potential anticancer efficacy of genistein‐topotecan combination in LNCaP prostate cancer cells and the mechanism of the combination treatment. The LNCaP cells were grown in complete RPMI medium, and cultured at 37°C, 5% CO₂ for 24–48 hrs to achieve 70–90% confluency. The cells were treated with varying concentrations of genistein, topotecan and genistein‐topotecan combination and incubated for 24 hrs. The treated cells were assayed for (i) post‐treatment sensitivity using MTT assay and DNA fragmentation, (ii) treatment‐induced apoptosis using caspase‐3 and ‐9 binding assays and (iii) treatment‐induced ROS generation levels. The overall data indicated that (i) both genistein and topotecan induce cellular death in LNCaP cells, (ii) genistein‐topotecan combination was significantly more efficacious in reducing LNCaP cell viability compared with either genistein or topotecan alone, (iii) in all cases, cell death was primarily through apoptosis, via the activation of caspase‐3 and ‐9, which are involved in the intrinsic pathway, (iv) ROS generation levels increased significantly with the genistein‐topotecan combination treatment. Treatments involving genistein‐topotecan combination may prove to be an attractive alternative phytotherapy or adjuvant therapy for prostate cancer.     

JS‐K induces reactive oxygen species‐dependent anti‐cancer effects by targeting mitochondria respiratory chain complexes in gastric cancer

As a nitric oxide (NO) donor prodrug, JS‐K inhibits cancer cell proliferation, induces the differentiation of human leukaemia cells, and triggers apoptotic cell death in various cancer models. However, the anti‐cancer effect of JS‐K in gastric cancer has not been reported. In this study, we found that JS‐K inhibited the proliferation of gastric cancer cells in vitro and in vivo and triggered mitochondrial apoptosis. Moreover, JS‐K induced a significant accumulation of reactive oxygen species (ROS), and the clearance of ROS by antioxidant reagents reversed JS‐K‐induced toxicity in gastric cancer cells and subcutaneous xenografts. Although JS‐K triggered significant NO release, NO scavenging had no effect on JS‐K‐induced toxicity in vivo and in vitro. Therefore, ROS, but not NO, mediated the anti‐cancer effects of JS‐K in gastric cancer. We also explored the potential mechanism of JS‐K‐induced ROS accumulation and found that JS‐K significantly down‐regulated the core proteins of mitochondria respiratory chain (MRC) complex I and IV, resulting in the reduction of MRC complex I and IV activity and the subsequent ROS production. Moreover, JS‐K inhibited the expression of antioxidant enzymes, including copper‐zinc‐containing superoxide dismutase (SOD1) and catalase, which contributed to the decrease of antioxidant enzymes activity and the subsequent inhibition of ROS clearance. Therefore, JS‐K may target MRC complex I and IV and antioxidant enzymes to exert ROS‐dependent anti‐cancer function, leading to the potential usage of JS‐K in the prevention and treatment of gastric cancer.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Effects of kinetin riboside on proliferation and proapoptotic activities in human normal and cancer cell lines

Kinetin riboside (KR) is a N6‐substituted derivative of adenosine. It is a natural compound which occurs in the milk of coconuts on the nanomole level. KR was initially shown to selectively inhibit proliferation of cancer cells and induce their apoptosis. We observed that KR inhibited growth (20–80%) of not only human cancer, but also normal cells and that this effect strongly depended on the type of cells. The anti‐apoptotic Bcl‐2 protein was downregulated, while proapoptotic Bax was upregulated in normal as well as in cancer cell lines, upon exposure to KR. Cytochrome c level increased in the cytosol upon treatment of cells with KR. The activity of caspases (ApoFluor®Green Caspase Activity Assay), as well as caspase‐3 (caspase‐3 activation assay) were increased mainly in cancer cells. The expression of procaspase 9 and its active form in the nucleus as well as in cytosol of KR‐treated cells was elevated. In contrast, no effect of KR on caspase 8 expression was noted. The results indicated that non‐malignant cells were less sensitive to KR then their cancer analogs and that KR most likely stimulated apoptosis mechanism of cancer cells through the intrinsic pathway. J. Cell. Biochem. 112: 2115–2124, 2011. © 2011 Wiley‐Liss, Inc.     

Reactive oxygen species‐induced cell death of rat primary astrocytes through mitochondria‐mediated mechanism

Astrocytes, the most abundant glial cell population in the central nervous system (CNS), play physiological roles in neuronal activities. Oxidative insult induced by the injury to the CNS causes neural cell death through extrinsic and intrinsic pathways. This study reports that reactive oxygen species (ROS) generated by exposure to the strong oxidizing agent, hexavalent chromium (Cr(VI)) as a chemical‐induced oxidative stress model, caused astrocytes to undergo an apoptosis‐like cell death through a caspase‐3‐independent mechanism. Although activating protein‐1 (AP‐1) and NF‐κB were activated in Cr(VI)‐primed astrocytes, the inhibition of their activity failed to increase astrocytic cell survival. The results further indicated that the reduction in mitochondrial membrane potential (MMP) was accompanied by an increase in the levels of ROS in Cr(VI)‐primed astrocytes. Moreover, pretreatment of astrocytes with N‐acetylcysteine (NAC), the potent ROS scavenger, attenuated ROS production and MMP loss in Cr(VI)‐primed astrocytes, and significantly increased the survival of astrocytes, implying that the elevated ROS disrupted the mitochondrial function to result in the reduction of astrocytic cell viability. In addition, the nuclear expression of apoptosis‐inducing factor (AIF) and endonuclease G (EndoG) was observed in Cr(VI)‐primed astrocytes. Taken together, evidence shows that astrocytic cell death occurs by ROS‐induced oxidative insult through a caspase‐3‐independent apoptotic mechanism involving the loss of MMP and an increase in the nuclear levels of mitochondrial pro‐apoptosis proteins (AIF/EndoG). This mitochondria‐mediated but caspase‐3‐independent apoptotic pathway may be involved in oxidative stress‐induced astrocytic cell death in the injured CNS. J. Cell. Biochem. 107: 933–943, 2009. © 2009 Wiley‐Liss, Inc.     

Localization of the anti‐cancer peptide EGFR‐lytic hybrid peptide in human pancreatic cancer BxPC‐3 cells by immunocytochemistry

Cationic lytic‐type peptides have been studied for clinical application in various infections and cancers, but their functional cellular mechanisms remain unclear. We generated anti‐cancer epithelial growth factor receptor (EGFR)‐lytic hybrid peptide, a 32‐amino‐acid peptide composed of an EGFR‐binding sequence and lytic sequence. In this study, we investigated the distribution of EGFR‐lytic hybrid peptide in BxPC‐3 human pancreatic cancer cells by an immunocytochemical (ICC) method. Distribution of EGFR protein expression was unchanged after treatment with EGFR‐lytic peptide compared with non‐treated cells. In confocal laser scanning microscopy, immunostaining of EGFR‐lytic peptide was observed in the cytoplasm, mostly in the form of granules. Some staining was also localized on the mitochondrial membrane. At the ultrastructure level, cells treated with EGFR‐lytic peptide had a low electron density, disappearance of microvilli, and swollen mitochondria. Fragments of cell membrane were also observed in the proximity of the membrane. In immunoelectron microscopy, EGFR‐lytic peptide was observed in the cell membrane and cytoplasm. A number of granules were considered swollen mitochondria. Activation of the caspase pathway as a result of mitochondrial dysfunction was also examined to determine the cytotoxic activity of EGFR‐lytic peptide; however, no effect on cell death after EGFR‐lytic treatment was observed, and moreover, apoptosis was not found to play a critical role in the cell death mechanism. These results suggest that EGFR‐lytic peptide is localized on cell and mitochondrial membranes, with disintegration of the cell membrane contributing mainly to cell death. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Induction of apoptosis by magnolol via the mitochondrial pathway and cell cycle arrest in renal carcinoma cells

Magnolol (Mag), an effective natural compound isolated from the stem bark of Magnolia officinalis, was found to have the potential for antitumor activity by inducing apoptosis in tumor cells. However, the effect of Mag on renal carcinoma cells and its molecular mechanism are unexplored. Our study provided evidence that Mag induced apoptosis in 786-O and OS-RC-2?cell lines via the mitochondrial pathway and cell cycle arrest. In this work, we found that Mag induced morphological changes and inhibited the proliferation of 786-O and OS-RC-2?cells in a dose- and time-dependent manner but exerted no notable inhibitory effects on normal human renal proximal tubular (HK-2) cells. Treatment with Mag suppressed the migration and invasion ability of renal carcinoma cells. Moreover, Mag caused the openness of mPTP, the accumulation of intracellular ROS and decreased △Ψm, leading to mitochondrial dysfunction. However, pretreatment with the antioxidant N-acetyl cysteine (NAC) reversed the apoptosis induced by Mag and decreased the generation of ROS. In addition, the increased proportion of the G1/G0 phase indicated that Mag caused cell cycle arrest. Further analyses suggested that magnolol-induced apoptosis was related to the abnormal expression of p53, Bax, Bcl-2, cytochrome c and caspase activation. Together, the results above revealed that Mag had antitumor effects in renal carcinoma cells via ROS production as well as cell cycle arrest and the apoptotic mitochondrial pathway was suppressed in part by NAC.     

Protection of neuronal cells against reactive oxygen species by carnosine and related compounds

Carnosine and related compounds were compared in terms of their abilities to decrease the levels of reactive oxygen species (ROS) in suspensions of isolated neurons activated by N-methyl-D-aspartic acid (NMDA) using both stationary fluorescence measurements and flow cytometry. Carnosine was found to suppress the fluorescent signal induced by ROS production and decreased the proportion of highly fluorescent neurons, while histidine showed opposite effects. N-Acetylated derivatives of both carnosine and histidine demonstrated weak (statistically indistinguishable) suppressive effects on the ROS signal. N-Methylated derivatives of carnosine suppressed intracellular ROS generation to the same extent as carnosine. This rank of effectiveness is distinct from that previously obtained for the anti-radical ability of CRCs (anserine>carnosine>ophidine). These differences suggest that the similar ability of carnosine and its N-methylated derivatives to protect neuronal cells against the excitotoxic effect of NMDA is not solely related to the antioxidant properties of these compounds.     

Targeting the ROS/PI3K/AKT/HIF‐1α/HK2 axis of breast cancer cells: Combined administration of Polydatin and 2‐Deoxy‐d‐glucose

It is well established that cancer cells depend upon aerobic glycolysis to provide the energy they need to survive and proliferate. However, anti‐glycolytic agents have yielded few positive results in human patients, in part due to dose‐limiting side effects. Here, we discovered the unexpected anti‐cancer efficacy of Polydatin (PD) combined with 2‐deoxy‐D‐glucose (2‐DG), which is a compound that inhibits glycolysis. We demonstrated in two breast cell lines (MCF‐7 and 4T1) that combination treatment with PD and 2‐DG induced cell apoptosis and inhibited cell proliferation, migration and invasion. Furthermore, we determined the mechanism of PD in synergy with 2‐DG, which decreased the intracellular reactive oxygen (ROS) levels and suppressed the PI3K/AKT pathway. In addition, the combined treatment inhibited the glycolytic phenotype through reducing the expression of HK2. HK2 deletion in breast cancer cells thus improved the anti‐cancer activity of 2‐DG. The combination treatment also resulted in significant tumour regression in the absence of significant morphologic changes in the heart, liver or kidney in vivo. In summary, our study demonstrates that PD synergised with 2‐DG to enhance its anti‐cancer efficacy by inhibiting the ROS/PI3K/AKT/HIF‐1α/HK2 signalling axis, providing a potential anti‐cancer strategy.     

A novel andrographolide derivative AL‐1 exerts its cytotoxicity on K562 cells through a ROS‐dependent mechanism

Andrographolide‐lipoic acid conjugate (AL‐1) is a new in‐house synthesized chemical entity, which was derived by covalently linking andrographolide with lipoic acid. However, its anti‐cancer effect and cytotoxic mechanism remains unknown. In this study, we found that AL‐1 could significantly inhibit cell viability of human leukemia K562 cells by inducing G2/M arrest and apoptosis in a dose‐dependent manner. Thirty‐one AL‐1‐regulated protein alterations were identified by proteomics analysis. Gene ontology and ingenuity pathway analysis revealed that a cluster of proteins of oxidative redox state and apoptotic cell death‐related proteins, such as PRDX2, PRDX3, PRDX6, TXNRD1, and GLRX3, were regulated by AL‐1. Functional studies confirmed that AL‐1 induced apoptosis of K562 cells through a ROS‐dependent mechanism, and anti‐oxidant, N‐acetyl‐l ‐cysteine, could completely block AL‐1‐induced cytotoxicity, implicating that ROS generation played a vital role in AL‐1 cytotoxicity. Accumulated ROS resulted in oxidative DNA damage and subsequent G2/M arrest and mitochondrial‐mediated apoptosis. The current work reveals that a novel andrographolide derivative AL‐1 exerts its anticancer cytotoxicity through a ROS‐dependent DNA damage and mitochondrial‐mediated apoptosis mechanism.     

Costunolide Induces Apoptosis through Generation of ROS and Activation of P53 in Human Esophageal Cancer Eca‐109 Cells

Costunolide is a sesquiterpene lactone, which possesses potent anti‐cancer properties. However, there is little report about its effects on esophageal cancer. In our study, we investigated the effects of costunolide on the cell viability, cell cycle, and apoptosis in human esophageal cancer Eca‐109 cells. It was found that costunolide inhibited the growth of Eca‐109 cells in a dose‐dependent manner, which was associated with the loss of mitochondrial membrane potential (Δψ_m) and the production of ROS. Costunolide induced apoptosis of Eca‐109 cells as well as cell cycle arrest in G1/S phase by upregulation of P53 and P21. Costunolide triggered apoptosis in esophageal cancer cells via the upregulation of Bax, downregulation of Bcl‐2, and significant activation of caspase‐3 and poly ADP‐ribose polymerase. These effects were markedly abrogated when cells were pretreated with N‐acetylcysteine, a specific reactive oxygen specie inhibitor. These results suggest that costunolide is a potential candidate for the treatment of esophageal cancer.