Molecular crosstalk between TRAIL and natural antioxidants in the treatment of cancer

The endogenous protein, tumour necrosis factor receptor apoptosis-inducing ligand (TRAIL), induces apoptosis in a wide variety of transformed and cancer cells but has little or no effect on normal cells. Therefore, TRAIL is considered to be a tumour-selective, apoptosis-inducing cytokine and a promising new candidate for cancer prevention and treatment. Some cancer cells are however resistant to TRAIL-induced apoptosis, but treatment in combination with conventional chemotherapeutic drugs or radiation generally restores TRAIL sensitivity in those cells. A novel class of molecules exhibiting synergy with TRAIL but devoid of major side effects are emerging as alternative approaches to treat resistant cancer cells, including natural antioxidants such as sulphoraphane or the flavonoids curcumin, quercetin, resveratrol, baicalein and wogonin. In this issue of the BJP, Lee et al. demonstrate that treatment of TRAIL-resistant cancer cells with wogonin restores TRAIL-induced cell death in a reactive oxygen species-dependent manner through up-regulation of p53 and Puma.     

Failure of Bay K 8644 to induce RhoA kinase-dependent calcium sensitization in rabbit blood vessels

Background and purpose:

Checkpoint kinase 2 (CHK2) is activated by DNA damage and can contribute to p53 stabilization, modulating growth arrest and/or apoptosis. We investigated the contribution of CHK2 to oxaliplatin-mediated toxicity in a colorectal cancer model.

Experimental approach:

We evaluated the ability of CHK2 small molecule inhibitors to potentiate oxaliplatin-induced toxicity. The role of CHK2 in oxaliplatin-induced apoptosis was investigated in HCT116 cells that were wild-type (WT) or KO for CHK2. Small molecule inhibitors of CHK2 were used in combination studies with oxaliplatin in this cell model.

Key results:

In oxaliplatin-treated CHK2 KO cells, accelerated apoptosis was accompanied by attenuated p53 stabilization and p21^WAF-1 up-regulation correlating with increased Bax expression, cytochrome c release and elevated caspase activity. The higher levels of apoptosis in CHK2 KO cells were restored to control (WT) levels when CHK2 was re-introduced. This ‘uncoupling’ of p53 stabilization and Bax up-regulation in CHK2 KO cells suggested oxaliplatin-induced apoptosis was due to a p53-independent response. Combination studies revealed that CHK2 inhibitor II or debromohymenialdisine antagonized the responses to oxaliplatin. This inhibitory effect correlated with decreases in apoptosis, p53 stabilization and DNA inter-strand cross-link formation, and was dependent on the presence (but not activity) of CHK2.

Conclusions and implications:

Combinations of CHK2 inhibitors with oxaliplatin should further sensitize cells to oxaliplatin treatment. However, these inhibitors produced an antagonistic effect on the response to oxaliplatin, which was reversed on the re-introduction of CHK2. These observations may have implications for the use of oxaliplatin in colorectal cancer therapy in combination with therapies targeting CHK2.     

Cardamonin sensitizes tumour cells to TRAIL through ROS- and CHOP-mediated up-regulation of death receptors and down-regulation of survival proteins

BACKGROUND AND PURPOSE

TNF-related apoptosis-inducing ligand (TRAIL) is currently in clinical trials as a treatment for cancer, but development of resistance is a major drawback. Thus agents that can overcome resistance to TRAIL are urgently needed. Cardamonin (2′,4′-dihydroxy-6′-methoxychalcone) has been shown to affect cell growth by modulating various cell signalling pathways. Hence, we investigated the effect of cardamonin on the actions of TRAIL.

EXPERIMENTAL APPROACH

The effect of cardamonin on TRAIL was measured by plasma membrane integrity, phosphatidylserine exposure, mitochondrial activity, and activation of caspase-8, caspase-9, and caspase-3 in human colon cancer cells.

KEY RESULTS

Cardamonin potentiated TRAIL-induced apoptosis and this correlated with up-regulation of both the TRAIL death receptor (DR) 4, 5 at mRNA and protein levels. TRAIL-decoy receptor DcR1 was down-regulated by cardamonin. Induction of DRs by cardamonin occurred in a variety of cell types. Gene silencing of the DRs by small interfering RNA (siRNA) abolished the effect of cardamonin on TRAIL-induced apoptosis, suggesting that sensitization was mediated through the DR. Induction of the DR by cardamonin was p53-independent but required CCAAT/enhancer binding protein homologous protein (CHOP); cardamonin induced CHOP, and its silencing by siRNA eliminated the induction of DR5. Cardamonin increased the production of reactive oxygen species (ROS) and quenching ROS abolished its induction of receptors and enhancement of TRAIL-induced apoptosis. Cardamonin also decreased the expression of various cell survival proteins.

CONCLUSIONS AND IMPLICATIONS

Cardamonin potentiates TRAIL-induced apoptosis through ROS-CHOP-mediated up-regulation of DRs, decreased expression of decoy receptor and cell survival proteins. Thus, cardamonin has the potential to make TRAIL more effective as an anticancer therapy.     

New benzimidazole acridine derivative induces human colon cancer cell apoptosis in vitro via the ROS-JNK signaling pathway

Aim:

To investigate the mechanisms underlying anticancer action of the benzimidazole acridine derivative N-{(1H-benzo[d]imidazol-2-yl)methyl}-2-butylacridin-9-amine(8m) against human colon cancer cells in vitro.

Methods:

Human colon cancer cell lines SW480 and HCT116 were incubated in the presence of 8m, and then the cell proliferation and apoptosis were measured. The expression of apoptotic/signaling genes and proteins was detected using RT-PCR and Western blotting. ROS generation and mitochondrial membrane depolarization were visualized with fluorescence microscopy.

Results:

8m dose-dependently suppressed the proliferation of SW480 and HCT116 cells with IC50 values of 6.77 and 3.33 μmol/L, respectively. 8m induced apoptosis of HCT116 cells, accompanied by down-regulation of Bcl-2, up-regulation of death receptor-5 (DR5), truncation of Bid, cleavage of PARP, and activation of caspases (including caspase-8 and caspase-9 as well as the downstream caspases-3 and caspase-7). Moreover, 8m selectively activated JNK and p38 without affecting ERK in HCT116 cells. Knockout of JNK1, but not p38, attenuated 8m-induced apoptosis. In addition, 8m induced ROS production and mitochondrial membrane depolarization in HCT116 cells. Pretreatment with the antioxidants N-acetyl cysteine or glutathione attenuated 8m-induced apoptosis and JNK activation in HCT116 cells.

Conclusion:

The new benzimidazole acridine derivative, 8m exerts anticancer activity against human colon cancer cells in vitro by inducing both intrinsic and extrinsic apoptosis pathways via the ROS-JNK1 pathway.     

Quercetin attenuates doxorubicin cardiotoxicity by modulating Bmi-1 expression

Background and Purpose

Doxorubicin-based chemotherapy induces cardiotoxicity, which limits its clinical application. We previously reported the protective effects of quercetin against doxorubicin-induced hepatotoxicity. In this study, we tested the effects of quercetin on the expression of Bmi-1, a protein regulating mitochondrial function and ROS generation, as a mechanism underlying quercetin-mediated protection against doxorubicin-induced cardiotoxicity.

Experimental Approach

Effects of quercetin on doxorubicin-induced cardiotoxicity was evaluated using H9c2 cardiomyocytes and C57BL/6 mice. Changes in apoptosis, mitochondrial function, oxidative stress and related signalling were evaluated in H9c2 cells. Cardiac function, serum enzyme activity and reactive oxygen species (ROS) generation were measured in mice after a single injection of doxorubicin with or without quercetin pre-treatment.

Key Results

In H9c2 cells, quercetin reduced doxorubicin-induced apoptosis, mitochondrial dysfunction, ROS generation and DNA double-strand breaks. The quercetin-mediated protection against doxorubicin toxicity was characterized by decreased expression of Bid, p53 and oxidase (p47 and Nox1) and by increased expression of Bcl-2 and Bmi-1. Bmi-1 siRNA abolished the protective effect of quercetin against doxorubicin-induced toxicity in H9c2 cells. Furthermore, quercetin protected mice from doxorubicin-induced cardiac dysfunction that was accompanied by reduced ROS levels and lipid peroxidation, but enhanced the expression of Bmi-1 and anti-oxidative superoxide dismutase.

Conclusions and Implications

Our results demonstrate that quercetin decreased doxorubicin-induced cardiotoxicity in vitro and in vivo by reducing oxidative stress by up-regulation of Bmi-1 expression. The findings presented in this study have potential applications in preventing doxorubicin-induced cardiomyopathy.     

Wogonin ameliorates lipotoxicity-induced apoptosis of cultured vascular smooth muscle cells via interfering with DAG-PKC pathway

Aim:

To investigate the effects of wogonin (5,7-dihydroxy-8-methoxyflavone) extracted from Scutellaria baicalensis Georgi (S baicalensis) on lipotoxicity-induced apoptosis of vascular smooth muscle cells (VSMCs) and the underlying mechanisms.

Methods:

Cultured VSMCs were used. Apoptosis of VSMCs was induced by palmitate (0.75 mmol/L), and detected using TUNEL assay. The expression levels of protein and phosphorylated protein were measured using Western blot analysis.

Results:

Treatment of VSMCs with wogonin (10, 25 and 50 μmol/L) significantly attenuated the apoptosis and endoplasmic reticulum (ER) stress induced by palmitate in concentration- and time-dependent manners. Wogonin (50 μmol/L) decreased palmitate-induced reactive oxygen species (ROS) generation. The ER stress inhibitor 4-phenyl butyric acid (5 mmol/L) significantly decreased palmitate-induced apoptotic cells, and occluded the anti-apoptotic effect of wogonin (25 μmol/L). Wogonin (10, 25 and 50 μmol/L) significantly reduced the intracellular diacylglycerol (DAG) accumulation and expression levels of phosphorylated PKCs in palmitate-treated VSMCs.

Conclusion:

Our results suggest that wogonin inhibits lipotoxicity-induced apoptosis of VSMCs via suppressing the intracellular DAG accumulation and subsequent inhibition of PKC phosphorylation. Wogonin has therapeutic potential for the prevention and treatment of atherosclerosis.     

Platinum-(IV)-derivative satraplatin induced G2/M cell cycle perturbation via p53-p21(waf1/cip1)-independent pathway in human colorectal cancer cells

Aim:

Platinum-(IV)-derivative satraplatin represents a new generation of orally available anti-cancer drugs that are under development for the treatment of several cancers. Understanding the mechanisms of cell cycle modulation and apoptosis is necessary to define the mode of action of satraplatin. In this study, we investigate the ability of satraplatin to induce cell cycle perturbation, clonogenicity loss and apoptosis in colorectal cancer (CRC) cells.

Methods:

CRC cells were treated with satraplatin, and the effects of satraplatin on apoptosis and the cell cycle were evaluated by flow cytometry. Western blot analysis was used to investigate the effects of satraplatin on cell cycle and apoptosis-related proteins. RT-qPCR was used to evaluate p53-related mRNA modulation.

Results:

Satraplatin induced an accumulation of CRC cells predominantly in the G₂/M phase. Increased p53 protein expression was observed in the p53 wild-type HCT116 and LoVo cells together with p21^waf1/cip1 protein up-regulation. However, p21^waf1/cip1 protein accumulation was not observed in the p53 mutant HCT15, HT29, and WiDr cells, even when p53 protein expression was compromised, suggesting that the cell cycle perturbation is p53-p21^waf1/cip1 independent. Following a candidate approach, we found an elevated expression of 14-3-3σ protein levels in CRC cells, which was independent of the status of p53, further supporting the role of satraplatin in the perturbation of the G₂/M cell cycle phase. Moreover, satraplatin treatment induced apoptosis along with Bcl-2 protein down-regulation and abrogated the clonogenic formation of CRC cells in vitro.

Conclusion:

Collectively, our data suggest that satraplatin induces apoptosis in CRC cells, which is preceded by cell cycle arrest at G₂/M due to the effect of 14-3-3σ and in a p53-p21^waf1/cip1–independent manner. Taken together, these findings highlight the potential use of satraplatin for CRC treatment.     

Salinomycin sensitizes cancer cells to the effects of doxorubicin and etoposide treatment by increasing DNA damage and reducing p21 protein

BACKGROUND AND PURPOSE

Salinomycin (Sal) has recently been shown to inhibit various cancer stem cells. Here, we investigated whether Sal could sensitize cancer cells to the effects of doxorubicin (DOX) or etoposide (ETO).

EXPERIMENTAL APPROACH

Using the Comet assay, immunocytochemistry and Western blot analysis, we assessed the ability of Sal to increase DNA breakage. We performed a cell proliferation assay to determine cell viability, cellular detachment, increased pre-G1 region, Annexin V staining and TUNEL assay to measure the ability of Sal to increase apoptosis.

KEY RESULTS

Sal increased DNA breakage and phosphorylated levels of p53 and H2AX. Sal also induced the formation of DNA foci with pH2AX and 53BP1. Furthermore, Sal increased the sensitivity of cancer cells to the apoptotic effects of DOX or ETO. We found that pH2AX, pBRCA1, p53BP1 and pChk1 levels were greatly increased after co-treatment of Sal with DOX or ETO. The level of anti-apoptotic p21 protein was increased by DOX or ETO but decreased by Sal, which increased proteasome activity.

CONCLUSIONS AND IMPLICATIONS

This is the first study to report that Sal increases DNA damage, and this effect plays an important role in the increased apoptosis caused by Sal. Overall, we demonstrated that the ability of Sal to sensitize cancer cells to the effects of DOX or ETO is associated with an increase in DNA damage and a decrease in anti-apoptotic protein p21 levels. These results may contribute to the development of Sal-based chemotherapy for cancer patients receiving DOX or ETO treatment.     

Bornyl caffeate induces apoptosis in human breast cancer cells in vitro via the ROS- and JNK-mediated pathways

Aim： To investigate the effects of bornyl caffeate discovered in several species of plant on human breast cancer cells in vitro and the underlying mechanisms.
Methods： Human breast cancer cell line MCF-7 and other tumor cell lines （T47D, HepG2, HeLa, and PC12） were tested. Cell viability was determined using MTT assay, and apoptosis was defined by monitoring the morphology of the nuclei and staining with Annexin V-FITC. Mitochondrial membrane potential （MMP） was measured using JC-1 under fluorescence microscopy. Intracellular reactive oxygen species （ROS） were assessed by flow cytometry. The expression of apoptosis-associated proteins was determined by Western blotting analysis.

Results： Bornyl caffeate （10, 25, and 50 μmol/L） suppressed the viability of MCF-7 cells in dose- and time-dependent manners, but neither caffeic acid nor borneol showed cytotoxicity at a concentration of 50 μmol/L. Bornyl caffeate also exerted cytotoxicity to HepG2, Hela, T47D, and PC12 cells. Bornyl caffeate dose-dependently induced apoptosis of MCF-7 cells, increased the expression of Bax and decreased the expression of Bcl-xl, resulting in the disruption of MMP and subsequent activation of caspase-3. Moreover, bornyl caffeate triggered the formation of ROS and activated p38 and c-Jun JNK. In MCF-7 cells, the cytotoxicity of bornyl caffeate was significantly attenuated by SB203580 （p38 inhibitor）, SP600125 （JNK inhibitor）, z-VAD （pan-caspase inhibitor） or the thiol antioxidant L-NAC.

Conclusion： Bornyl caffeate exerts non-selective cytotoxicity against cancer cells of different origin in vitro. The compound induces apoptosis in human breast cancer MCF-7 cells via the ROS- and JNK-mediated pathways.     

Anticancer bioactive peptides suppress human colorectal tumor cell growth and induce apoptosis via modulating the PARP-p53-Mcl-1 signaling pathway

Aim:

We have reported novel anticancer bioactive peptides (ACBPs) that show tumor-suppressive activities in human gastric cancer, leukemia, nasopharyngeal cancer, and gallbladder cancer. In this study, we investigated the effects of ACBPs on human colorectal cancer and the underlying mechanisms.

Methods:

Cell growth and apoptosis of human colorectal tumor cell line HCT116 were measured using cell proliferation assay and flow cytometry, respectively. The expression levels of PARP, p53 and Mcl1A were assessed with Western blotting and immunohistochemistry. For evaluation of the in vivo antitumor activity of ACBPs, HCT116 xenograft nude mice were treated with ACBPs (35 μg/mL, ip) for 10 days.

Results:

Treatment of HCT116 cells with ACBPs (35 μg/mL) for 4–6 days significantly inhibited the cell growth. Furthermore, treatment of HCT116 cells with ACBPs (35 μg/mL) for 6–12 h significantly enhanced UV-induced apoptosis, increased the expression of PARP and p53, and decreased the expression of Mcl-1. Administration of ACBPs did not change the body weight of HCT116 xenograft nude mice, but decreased the tumor growth by approximately 43%, and increased the expression of PARP and p53, and decreased the expression of Mcl-1 in xenograft mouse tumor tissues.

Conclusion:

Administration of ACBPs inhibits human colorectal tumor cell growth and induces apoptosis in vitro and in vivo through modulating the PARP-p53-Mcl-1 signaling pathway.     

Telekin suppresses human hepatocellular carcinoma cells in vitro by inducing G2/M phase arrest via the p38 MAPK signaling pathway

Aim:

Telekin, isolated from the Chinese herb Carpesium divaricatum, has shown anti-proliferation effects against various cancer cells, including hepatocellular carcinoma cells. In this study, we investigated the anti-proliferation mechanisms of telekin in human hepatocellular carcinoma HepG2 cells in vitro.

Methods:

HepG2 cells were treated with telekin. Cell viability was evaluated using MTT assay. Flow cytometry was used to measure cell cycle profiles, ROS level and apoptosis. The protein expression levels were analyzed with Western blotting.

Results:

Telekin (3.75–30 μmol/L) dose-dependently inhibited the viability of HepG2 cells and induced l apoptosis. Furthermore, the treatment induced cell cycle arrest at G₂/M phase, accompanied by significantly increased the phosphorylation of Cdc25A and Cdc2, and decreased Cyclin B1 level. Moreover, the treatment significantly stimulated ROS production, and increased the phosphorylation of p38 and MAPKAPK-2 in the cells. Pretreatment with the antioxidant NAC (2.5, 5, and 10 mmol/L), or the p38 MAPK inhibitor SB203580 (2.5 and 5 μmol/L) dose-dependently attenuated these telekin-induced effects in the cells.

Conclusion:

Telekin suppresses hepatocellular carcinoma cells in vitro by inducing G₂/M phase arrest via activating the p38 MAPK pathway.     

Silencing Prx1 and/or Prx5 sensitizes human esophageal cancer cells to ionizing radiation and increases apoptosis via intracellular ROS accumulation

Aim:

To investigate whether down-regulation of peroxiredoxin 1 (Prx1) and/or peroxiredoxin 5 (Prx5) sensitizes human esophageal cancer cells to ionizing radiation (IR).

Methods:

Human esophageal carcinoma cell lines Eca-109 and TE-1 were used. Prx mRNA expression profiles in Eca-109 and TE-1 cells were determined using RT-PCR. Two highly expressed isoforms of Prxs, Prx1 and Prx5, were silenced by RNA interference (RNAi). Following IR, intracellular reactive oxygen species (ROS) and apoptosis were measured using flow cytometry, the activities of catalase, superoxide dismutase and glutathione peroxidase were measured, and the radiosensitizing effect of RNAi was observed. Tumor xenograft model was also used to examine the radiosensitizing effect of RNAi in vivo.

Results:

Down-regulation of Prx1 and/or Prx5 by RNAi does not alter the activities of catalase, superoxide dismutase and glutathione peroxidase, but made human tumor cells more sensitive to IR-induced apoptosis both in vitro and in vivo. When the two isoforms were decreased simultaneously, intracellular ROS and apoptosis significantly increased after IR.

Conclusion:

Silencing Prx1 and/or Prx5 by RNAi sensitizes human Eca-109 and TE-1 cells to IR, and the intracellular ROS accumulation may contribute to the radiosensitizing effect of the RNAi.     

RIP3 overexpression sensitizes human breast cancer cells to parthenolide in vitro via intracellular ROS accumulation

Aim： Receptor-interacting protein 3 （RIP3） is involved in tumor necrosis factor receptor signaling, and results in NF-KB-mediated prosurvival signaling and programmed cell death. The aim of this study was to determine whether overexpression of the RIP3 gene could sensitize human breast cancer cells to parthenolide in vitro. Methods： The expression of RIP3 mRNA in human breast cancer cell lines （MCF-7, MDA-MB-231, MDA-MB-435 and T47D） was detected using RT-PCR. Both MDA-MB-231 and MCF-7 cells were transfected with RIP3 expression or blank vectors via lentivirus. Cell viability was measured with MTT assay; intracellular ROS level and cell apoptosis were analyzed using flow cytometry. Results： RIP3 mRNA expression was not detected in the four human breast cancer cell lines tested. However, the transfection induced higher levels of RIP3 protein in MCF-7 and MDA-MB-231 cells. Furthermore, overexpression of RIP3 decreased the IC50 values of parthenolide from 17.6 to 12.6 μmol/L in MCF-7 cells, and from 16.6 to 9.9 μmol/L in MDA-MB-231 cells. Moreover, overexpression of RIP3 significantly increased parthenolide-induced apoptosis and ROS accumulation in MCF-7 and MDA-MB-231 cells. Pretreatment with N-acetyl-cysteine abrogated the increased sensitivity of RIP3-transfected MCF-7 and MDA-MB-231 cells to parthenolide. Conclusion： Overexpression of RIP3 sensitizes MCF-7 and MDA-MB-231 breast cancer cells to parthenolide in vitro via intracellular ROS accumulation.     

In rat renal fibroblasts, mycophenolic acid inhibits proliferation and production of the chemokine CCL2, stimulated by tumour necrosis factor-��

BACKGROUND AND PURPOSE

Renal fibroblasts play a pivotal role in the development of tubulointerstitial fibrosis, a condition highly predictive of progression towards end-stage renal disease. The present study investigated the anti-mitogenic and anti-inflammatory effects of an inhibitor of inosine monophosphate dehydrogenase, mycophenolic acid (MPA) and the mechanisms underlying its action in normal rat kidney fibroblasts (49F cells).

EXPERIMENTAL APPROACH

Proliferation of 49F cells was studied by tetrazole 3-(4, 5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT) test, bromodeoxyuridine incorporation and flow cytometry. The cyclins, tumour suppressor genes and phospho-mitogen-activated protein kinases (MAPKs) were semiquantified by immunoblotting. Apoptosis was measured by quantifying the fragmented DNA and the activity of caspase 3. The monocyte chemokine CCL2 was measured by ELISA. The mRNA expression of CCL2 was measured by real-time PCR.

KEY RESULTS

Mycophenolic acid dose-dependently inhibited steady-state proliferation of 49F cells by up-regulation of p21, p27 and p53, in association with a decrease in cyclins D2 and E. Treatment with MPA also triggered apoptosis of 49F cells by activating the caspase 3 cascade. Furthermore, MPA attenuated tumour necrosis factor-α-induced CCL2 expression through down-regulation of p38 MAPK, but not that of ERK1/2 or JNK.

CONCLUSIONS AND IMPLICATIONS

The anti-mitogenic and anti-inflammatory effects of MPA were mediated by up-regulation of cell cycle inhibitors and pro-apoptotic signals, and by suppression of p38 MAPK pathway respectively. This dual effect of MPA may form the rationale for animal or clinical trials for the treatment of fibrotic renal diseases.     

Physalin B not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in human colon cancer cells in vitro

Aim:

To investigate the effects of physalin B insolated from Physalis divericata on human colon cancer cells in vitro and its anticancer mechanisms.

Methods:

Human HCT116 colon cancer cell line was tested. Cell viability and apoptosis were detected, and relevant proteins were measured using Western blot analyses. Autophagosomes were observed in stable GFP-LC3 HCT116 cells. Localization of autophagosomes and lysosomes was evaluated in GFP-LC3/RFP-LAMP1-co-transfected cells. Microtubules and F-actin microfilaments were observed with confocal microscope. Mitochondrial ROS (mito-ROS) was detected with flow cytometry in the cells stained with MitoSox dye.

Results:

Physalin B inhibited the viability of HCT116 cells with an IC₅₀ value of 1.35 μmol/L. Treatment of the cells with physalin B (2.5–10 μmol/L) induced apoptosis and the cleavage of PARP and caspase-3. Meanwhile, physalin B treatment induced autophagosome formation, and accumulation of LC3-II and p62, but decreased Beclin 1 protein level. Marked changes of microtubules and F-actin microfilaments were observed in physalin B-treated cells, which led to the blockage of co-localization of autophagosomes and lysosomes. Physalin B treatment dose-dependently increased the phosphorylation of p38, ERK and JNK in the cells, whereas the p38 inhibitor SB202190, ERK inhibitor U0126 or JNK inhibitor SP600125 could partially reduce physalin B-induced PARP cleavage and p62 accumulation. Moreover, physalin B treatment dose-dependently increased mito-ROS production in the cells, whereas the ROS scavenger NAC could reverse physalin B-induced effects, including incomplete autophagic response, accumulation of ubiquitinated proteins, changes of microtubules and F-actin, activation of p38, ERK and JNK, as well as cell death and apoptosis.

Conclusion:

Physalin B induces mito-ROS, which not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in HCT116 cells in vitro.     

G226, a new epipolythiodioxopiperazine derivative,triggers DNA damage and apoptosis in human cancer cells in vitro via ROS generation

Aim:

G226 is a novel derivative of epipolythiodioxopiperazines with potent inhibitory activity against cancer cells. Here, we sought to identify potential targets involved in the anti-cancer activity of G226.

Methods:

Cell proliferation assay was conducted in a panel of 12 human cancer cell lines. The activities of topoisomerase I (Topo I) and Topo II were studied using supercoiled pBR322 DNA relaxation and kDNA decatenation assays. ROS production was assessed with probes DCFH-DA and H&E. Western blot analysis and flow cytometry were used to examine DNA damage, apoptosis and cell cycle changes.

Results:

G226 displayed potent cytotoxicity in the 12 human cancer cell lines with a mean IC₅₀ value of 92.7 nmol/L. This compound (1–100 μmol/L) selectively inhibited the activity of Topo II, and elevated the expression of phosphorylated-H2AX in a dose-dependent manner. In Topo II-deficient HL60/MX2 cells, however, G226-induced DNA damage, apoptosis and cytotoxicity were only partially reduced, suggesting that Topo II was not essential for the anti-tumor effects of G226. Furthermore, G226 (0.125–2 μmol/L) dose-dependently elevated the intracellular levels of H₂O₂ and in the cancer cells, and pretreatment with GSH, NAC or DTT not only blocked G226-induced intracellular accumulation of ROS, but also abrogated G226-mediated phosphorylation of H2AX, apoptosis and cytotoxicity.

Conclusion:

G226-mediated ROS production contributes to the anti-cancer activity of this compound.     

Cathepsin L suppression increases the radiosensitivity of human glioma U251 cells via G2/M cell cycle arrest and DNA damage

Aim:

Cathepsin L is a lysosomal cysteine protease that plays important roles in cancer tumorigenesis, proliferation and chemotherapy resistance. The aim of this study was to determine how cathepsin L regulated the radiosensitivity of human glioma cells in vitro.

Methods:

Human glioma U251 cells (harboring the mutant type p53 gene) and U87 cells (harboring the wide type p53 gene) were irradiated with X-rays. The expression of cathepsin L was analyzed using Western blot and immunofluorescence assays. Cell survival and DNA damage were evaluated using clonogenic and comet assays, respectively. Flow cytometry was used to detect the cell cycle distribution. Apoptotic cells were observed using Hoechst 33258 staining and fluorescence microscopy.

Results:

Irradiation significantly increased the cytoplasmic and nuclear levels of cathepsin L in U251 cells but not in U87 cells. Treatment with the specific cathepsin L inhibitor Z-FY-CHO (10 μmol/L) or transfection with cathepsin L shRNA significantly increased the radiosensitivity of U251 cells. Both suppression and knockdown of cathepsin L in U251 cells increased irradiation-induced DNA damage and G₂/M phase cell cycle arrest. Both suppression and knockdown of cathepsin L in U251 cells also increased irradiation-induced apoptosis, as shown by the increased levels of Bax and decreased levels of Bcl-2.

Conclusion:

Cathepsin L is involved in modulation of radiosensitivity in human glioma U251 cells (harboring the mutant type p53 gene) in vitro.     

Crocetin induces cytotoxicity and enhances vincristine-induced cancer cell death via p53-dependent and -independent mechanisms

Aim:

To investigate the anticancer effect of crocetin, a major ingredient in saffron, and its underlying mechanisms.

Methods:

Cervical cancer cell line HeLa, non-small cell lung cancer cell line A549 and ovarian cancer cell line SKOV3 were treated with crocetin alone or in combination with vincristine. Cell proliferation was examined using MTT assay. Cell cycle distribution and sub-G₁ fraction were analyzed using flow cytometric analysis after propidium iodide staining. Apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit with flow cytometry. Cell death was measured based on the release of lactate dehydrogenase (LDH). The expression levels of p53 and p21^WAF1/Cip1 as well as caspase activation were examined using Western blot analysis.

Results:

Treatment of the 3 types of cancer cells with crocetin (60-240 μmol/L) for 48 h significantly inhibited their proliferation in a concentration-dependent manner. Crocetin (240 μmol/L) significantly induced cell cycle arrest through p53-dependent and -independent mechanisms accompanied with p21^WAF1/Cip1 induction. Crocetin (120-240 μmol/L) caused cytotoxicity in the 3 types of cancer cells by enhancing apoptosis in a time-dependent manner. In the 3 types of cancer cells, crocetin (60 μmol/L) significantly enhanced the cytotoxicity induced by vincristine (1 μmol/L). Furthermore, this synergistic effect was also detected in the vincristine-resistant breast cancer cell line MCF-7/VCR.

Conclusion:

Ccrocetin is a potential anticancer agent, which may be used as a chemotherapeutic drug or as a chemosensitizer for vincristine.     

Resveratrol analogue 3,4,4′-trihydroxy-trans-stilbene induces apoptosis and autophagy in human non-small-cell lung cancer cells in vitro

Aim:

To investigate the effects of 3,4,4′-trihydroxy-trans-stilbene (3,4,4′-THS), an analogue of resveratrol, on human non-small-cell lung cancer (NSCLC) cells in vitro.

Methods:

Cell viability of NSCLC A549 cells was determined by MTT assay. Cell apoptosis was evaluated using flow cytometry and TUNEL assay. Cell necrosis was evaluated with LDH assay. The expression of apoptosis- or autophagy-associated proteins was measured using Western blotting. The formation of acidic compartments was detected using AO staining, neutral red staining and Lysotracker-Red staining. LC3 punctae were analyzed with fluorescence microscopy.

Results:

Treatment with 3,4,4′-THS (10-80 μmol/L) concentration-dependently inhibited the cell viability. It did not cause cell necrosis, but induced apoptosis accompanied by up-regulation of cleavaged PARP, caspase3/9 and Bax, and by down-regulation of Bcl-2 and surviving. It also increased the formation of acidic compartments, LC3-II accumulation and GFP-LC3 labeled autophagosomes in the cells. It inhibited the mTOR-dependent pathway, but did not impair autophagic flux. 3,4,4′-THS-induced cell death was enhanced by the autophagy inhibitors 3-MA (5 mmol/L) or Wortmannin (2 μmol/L). Moreover, 3,4,4′-THS treatment elevated the ROS levels in the cells, and co-treatment with 3-MA further elevated the ROS levels. 3,4,4′-THS-induced apoptosis and autophagy in the cells was attenuated by NAC (10 mmol/L)

Conclusion:

3,4,4′-THS induces both apoptosis and autophagy in NSCLC A549 cells in vitro. Autophagy inhibitors promote 3,4,4′-THS-induced apoptosis of A549 cells, thus combination of 3,4,4′-THS and autophagy inhibitor provides a promising strategy for NSCLC treatment.