Retigeric acid B-induced mitophagy by oxidative stress attenuates cell death against prostate cancer cells in vitro

Aim:

Retigeric acid B (RAB), a pentacyclic triterpenic acid from Lobaria kurokawae Yoshim, has been found to induce apoptosis in prostate cancer cells. The aim of this study was to investigate the roles of mitochondrial damage-caused mitophagy in RAB-induced prostate cancer cell death in vitro.

Methods:

Human prostate cancer PC3 and LNCaP cells were tested. Cell viability was analyzed with MTT assay. Cell apoptosis, ROS level and mitochondrial transmembrane potential (mtΔψ) were measured with flow cytometry. Autophagy- and apoptosis-related proteins were studied using Western blotting. GFP-LC3B puncta, mitochondrial swelling and mitophagy were examined morphologically. Quantitative RT-PCR was used to measure LC3B mRNA level, and siRNA was used to knock down LC3BII.

Results:

In both PC3 and LNCaP cells, RAB (15 μmol/L) increased ROS accumulation and decreased mtΔψ in a time-dependent manner. Furthermore, RAB induced mitochondrial swelling and mitophagy, significantly increased LC3B expression and conversion of LC3BI to LC3BII, and the elimination of mitochondria by LC3BII-containing autophagolysosomes. In addition, RAB suppressed the PI3K/Akt/mTOR pathway activation. Pretreatment of PC3 cells with autophagy inhibitor 3-MA (5 mmol/L) or the lysosomal protease inhibitor CQ (10 μmol/L) significantly increased RAB-induced apoptosis. Similar results were obtained in RAB-treated PC3 cells with LC3B knocked down.

Conclusion:

RAB induces mitochondrial damage and mitophagy that attenuates RAB-induced prostate cancer cell death. Thus, suppression of mitophagy might be a potential strategy for improving the chemotherapeutic effects of RAB.     

Genome expression analysis by suppression subtractive hybridization identified overexpression of Humanin, a target gene in gastric cancer chemoresistance

Background

In cancer cells, apoptosis is an important mechanism that influences the outcome of chemotherapy and the development of chemoresistance. To find the genes involved in chemoresistance and the development of gastric cancer, we used the suppression subtractive hybridization method to identify the genes that are overexpressed in gastric cancer tissues compared to normal gastric tissues.

Results

In the suppression subtractive hybridization library we constructed, the most highly overexpressed genes were humanin isoforms. Humanin is a recently identified endogenous peptide that has anti-apoptotic activity and has been selected for further study due to its potential role in the chemoresistance of gastric cancer. Upregulation of humanin isoforms was also observed in clinical samples by using quantitative real-time PCR. Among the studied isoforms, humanin isoform 3, with an expression level of 4.166 ± 1.44 fold, was the most overexpressed isoform in GC.

Conclusions

The overexpression of humanin in gastric cancer suggests a role for chemoresistance and provides new insight into the biology of gastric cancer. We propose that humanin isoforms are novel targets for combating chemoresistance in gastric cancer.     

Cyclic bisbibenzyls induce growth arrest and apoptosis of human prostate cancer PC3 cells

Aim:

To investigate the cytotoxic effects of four cyclic bisbibenzyls, Riccardin C (Ric), Pakyonol (Pak), Marchantin M (Mar), and Plagiochin E (Pla) against chemoresistant prostate cancer PC3 cells.

Methods:

Cell growth was assayed by MTT method, and apoptotic related protein Bcl-2 and Bax, poly(ADP-ribose) polymerase (PARP) were examined by Western blotting. Cell cycle and apoptosis of PC3 cells were evaluated with flow cytometry and morphologic examinations.

Results:

The four compounds inhibited proliferation and elicited cell death in a dose- and time-dependent manner with IC₅₀ values of 3.22 μmol/L for Ric, 7.98 μmol/L for Pak, 5.45 μmol/L for Mar, and 5.99 μmol/L for Pla, respectively. Furthermore, exposed to these chemicals caused a decrease in the antiapoptotic protein Bcl-2 and an increase in proapoptotic Bax expression. PARP cleavage and caspase-3 activity were also observed.

Conclusion:

The results suggest that cyclic bisbibenzyls could be used for the development of novel therapeutic chemicals against prostate cancer.     

GLP-1 receptor plays a critical role in geniposideinduced expression of heme oxygenase-1 in PC12 cells

Aim:

To explore the role of activation of glucagon-like peptide 1 receptor (GLP-1R) and its relative cell signaling pathway in the cytoprotection of geniposide.

Methods:

Cell viability was determined by MTT assay. Knockdown of the Glp-1r gene was carried out with shRNA. The levels of HO-1 protein and cAMP response element binding protein (CREB) phosphorylation were measured by Western blotting.

Results:

Geniposide protected PC12 cells from oxidative damage induced by 3-morpholinosydnonimine hydrochloride (SIN-1) by enhancing the expression of heme oxygenase 1 (HO-1) via the cAMP-PKA-CREB signal pathway. After transfecting PC12 cells with the AB1 enhancer from the HO-1 gene, luciferase activity induced by geniposide increased in a dose-dependent manner, but not in the PC12 cells whose Glp-1r gene was disrupted. Additionally, inhibition of HO-1 activity by Sn-protoporphyrin IX (SnPP) or shRNA-mediated knockdown of Glp-1r decreased the neuroprotection of geniposide in PC12 cells.

Conclusion:

GLP-1R plays a critical role in geniposide-induced HO-1 expression to attenuate oxidative insults in PC12 cells.     

Sesquiterpenoids from myrrh inhibit androgen receptor expression and function in human prostate cancer cells

Aim:

To examine whether two naturally occurring sesquiterpenoids (ST1 and ST2) with anti-proliferative activity in prostate cancer cells inhibit androgen receptor (AR) signaling.

Methods:

Human prostate cancer cell lines LNCaP and PC3 were used. The expression of AR, AR translocation into the nucleus, and expression levels of AR coactivators ARA70 and steroid receptor coactivator-1 (SRC-1) in LNCaP cells were examined using real-time PCR and Western blot. Changes in prostate-specific antigen (PSA) protein levels, PSA promoter activity, and androgen response element (ARE)-mediated reporter gene activity were examined using enzyme-linked immunoabsorbent assay (ELISA) and transient transfection assays. Co-immunoprecipitation was performed to analyze the interaction between AR and the AR coactivators in ST1- and ST2-treated cells.

Results:

In LNCaP cells, ST1 and ST2 (40 μmol/L) led to a significant decrease in the expression of AR as well as a reduction of AR translocation into the nucleus, but had no effect on AR protein translation. ST1 and ST2 treatment also resulted in a significant decrease in the level of PSA protein secreted into the medium and was able to suppress PSA promoter-dependent and ARE-dependent luciferase activity. Furthermore, decreased expression of ARA70 and SRC-1 was observed when LNCaP cells were exposed to ST1 and ST2, which interfered with their ability to interact with AR.

Conclusion:

The observations suggest that suppression of AR transactivation by ST1 and ST2 may be mediated, in part, by inhibiting AR nuclear translocation and/or interfering with the interaction between AR and its coactivators ARA70 and SRC-1. Therefore, sesquiterpenoids could be developed as novel therapeutic agents for treating prostate cancer.     

Paeonol protects rat vascular endothelial cells from ox-LDL-induced injury in vitro via downregulating microRNA-21 expression and TNF-α release

Aim： Paeonol （2＇-hydroxy-4＇-methoxyacetophenone） from Cortex moutan root is a potential therapeutic agent for atherosclerosis. This study sought to investigate the mechanisms underlying anti-inflammatory effects of paeonol in rat vascular endothelial cells （VECs） in vitro.
Methods： VECs were isolated from rat thoracic aortas. The cells were pretreated with paeonol for 24 h, and then stimulated with ox-LDL for another 24 h. The expression of microRNA-21 （miR-21） and PTEN in VECs was analyzed using qRT-PCR. The expression of PTEN protein was detected by Western blotting. TNF-α release by VECs was measured by ELISA.

Results： Ox-LDL treatment inhibited VEC growth in dose- and time-dependent manners （the value of IC50 was about 20 mg/L at 24 h）. Furthermore, ox-LDL （20 mg/L） significantly increased miR-21 expression and inhibited the expression of PTEN, one of downstream target genes of miR-21 in VECs. In addition, ox-LDL （20 mg/L） significantly increased the release of TNF-α from VECs. Pretreatment with paeonol increased the survival rate of ox-LDL-treated VECs in dose- and time-dependent manners. Moreover, paeonol （120 μmol/L） prevented ox-LDL-induced increases in miR-21 expression and TNF-α release, and ox-LDL-induced inhibition in PTEN expression. A dual-luciferase reporter assay showed that miR-21 bound directly to PTEN＇s 3＇-UTR, thus inhibiting PTEN expression. In ox-LDL treated VECs, transfection with a miR-21 mimic significantly increased miR-21 expression and inhibited PTEN expression, and attenuated the protective effects of paeonol pretreatment, whereas transfection with an miR-21 inhibitor significantly decreased miR-21 expression and increased PTEN expression, thus enhanced the protective effects of paeonol pretreatment.

Conclusion： miR-21 is an important target of paeonol for its protective effects against ox-LDL-induced VEC injury, which may play critical roles in development of atherosclerosis.     

Hsa-miR-520d Converts Fibroblasts into CD105+ Populations

Background

We have previously shown that hsa-miR-520d-5p can convert cancer cells into induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (MSCs) via a dedifferentiation by a demethylation mechanism.

Methods

We tested the effect of miR-520d-5p on human fibroblasts to determine whether it could be safely used in normal cells for future clinical therapeutic applications. After we transfected the microRNA into fibroblasts, we analyzed the phenotypic changes, gene expression levels, and stemness induction in vitro, and we evaluated tumor formation in an in vivo xenograft model.

Results

The transfected fibroblasts turned into CD105+ cell populations, survived approximately 24 weeks, and exhibited increases in both the collagen-producing ability and in differentiation. Combinatorial transfection of small interfering RNAs for miR-520d-5p target genes (ELAVL2, GATAD2B, and TEAD1) produced similar results to miR-520d-5p transfection. These molecules converted normal cells into MSCs and not iPSCs.

Conclusions

In vitro data indicate the potent usefulness of this small molecule as a therapeutic biomaterial in normal cells and cancer cells because CD105+ cells never converted to iPSCs despite repeated transfections and all types of transfectants lost their tumorigenicity. This maintenance of a benign state following miR-520d-5p transfection appears to be caused by p53 upregulation. We conclude that miR-520d-5p may be a useful biomaterial at an in vitro level.

Electronic supplementary material

The online version of this article (doi:10.1007/s40268-014-0064-6) contains supplementary material, which is available to authorized users.     

Methyl jasmonate sensitizes human bladder cancer cells to gambogic acid-induced apoptosis through down-regulation of EZH2 expression by miR-101

Background and Purpose

Gambogic acid (GA) and methyl jasmonate (MJ) are increasingly being recognized as novel natural anticancer compounds. Here, we investigated the antitumour effects of GA in combination with MJ on human bladder cancer cells.

Experimental Approach

Cell viability was detected by cell counting kit-8 assay. Cell apoptosis was assessed by Hoechst 33258 staining and flow cytometry. Protein levels were determined by immunoblotting and expressions of mRNA and miRNAs by RT-PCR. Differential expressions of a group of downstream genes were identified using microarray analysis.

Key Results

MJ significantly sensitized bladder cancer cells to GA-induced growth inhibition and apoptosis while sparing normal fibroblasts. MJ enhanced GA-induced activation of caspase-3 and caspase-9, and down-regulated the expression of XIAP. Furthermore, treatment of bladder cancer cells with a combination of GA and MJ induced synergistic inhibition of the enhancer of zeste homologue 2 (EZH2) expression, whereas miR-101 expression was up-regulated. Conversely, knockdown of miR-101 restored this decreased expression of EZH2 and suppressed the inhibitory effect of GA and MJ on the growth of bladder cancer cells. Microarray analysis showed that genes closely associated with bladder cancer development were significantly down-regulated by GA and MJ. In a s.c. xenograft mouse model of human bladder carcinoma, the combination of GA and MJ exerted an increased antitumour effect compared with GA alone.

Conclusion and Implications

MJ sensitizes bladder cancer cells to GA-induced apoptosis by down-regulating the expression of EZH2 induced by miR-101. Thus, the combination of selective anti-cancer agents MJ and GA could provide a novel strategy for treating human bladder cancer.     

Development of β-amino-carbonyl compounds as androgen receptor antagonists

Aim： Androgen receptor （AR） antagonists have proven to be useful in the early control of prostate cancer. The aim of this study was to identify and characterize a novel β-amino-carbonyl-based androgen receptor antagonist. Methods Different isomers of the β-amino-carbonyl compounds were obtained by chiral separation. The bioactivities of the isomers were evaluated by AR nuclear translocation, mammalian two-hybrid, competitive receptor binding and cell proliferation assays. The expression of genes downstream of AR was analyzed with real-time PCR. The therapeutic effects on tumor growth in vivo were observed in male SClD mice bearing LNCaP xenografts. Results： Compound 21 was previously identified as an AR modulator by the high-throughput screening of a diverse compound library. In the present study, the two isomers of compound 21, termed compounds 21-1 and 2.1.-2, were characterized as partial AR agonists in terms of androgen-induced AR nuclear translocation, prostate-specific antigen expression and cell proliferation. Further structural modifications led to the discovery of a androgen receptor antagonist （compound 6012）, which blocked androgen receptor nuclear translocation, androgen-responsive gene expression and androgen-dependent LNCaP cell proliferation. Four stereoisomers of compound 6012 were isolated, and their bioactivities were assessed. The pharmacological effects of 6012, including AR binding, androgen-induced AR translocation, NH2- and COOH-terminal interaction, growth inhibition of LNCaP cells in vitro and LNCaP xenograft growth in nude mice, were mainly restricted to isomer 6012-4 （1R, 3S）. Conclusion： Compound 6012-4 was determined to be a novel androgen receptor antagonist with prostate cancer inhibitory activities comparable to bicalutamide both in vitro and in vivo.     

Construction of lentivirus-based inhibitor of hsa-microRNA-338-3p with specific secondary structure

Aim:

To construct a lentivirus-based inhibitor with specific secondary structure that could exert long-term suppression on microRNA-338-3p (miR-338-3p), thus elucidating its molecular function in colorectal carcinoma cells.

Methods:

The miR-338-3p inhibitor sequence was synthesized and inserted into pLV-THM plasmid. HEK-293T cells were co-transfected with the lentiviral vectors pLV-THM-miR-338-3p-inhibitor, psPAX2, and pMD2.G. The supernatant containing the lentivirus particles was harvested to determine the viral titer, and then used to infect colorectal carcinoma-derived SW-620 cells. eGFP(+) cells were sorted using flow cytometry. The expression of miR-338-3p in SW-620 cells was determined with real-time RT-PCR, and the expression of the smoothened (SMO) protein was detected using Western blot analysis. The migration ability of the transfected SW-620 cells was assessed with transwell assay.

Results:

Restriction endonuclease analysis and DNA sequencing demonstrated that the lentiviral vector pLV-THM-miR-338-3p-inhibitor was successfully constructed. The expression of miR-338-3p in SW-620 cells was significantly decreased by infection with the lentivirus pLV-THM-miR-338-3p-inhibitor. Moreover, the down-regulated expression of miR-338-3p caused up-regulated expression of the SMO protein in SW-620 cells, which showed significantly enhanced migration in transwell assay.

Conclusion:

The construction of the lentiviral vector pLV-THM-miR-338-3p-inhibitor with specific secondary structure provides a basis for further studies the molecular function of miR-338-3p in colorectal carcinoma. miR-338-3p may suppress SMO gene expression and thereby inhibit colorectal carcinoma migration.     

Reactive oxygen species up-regulate p53 and Puma; a possible mechanism for apoptosis during combined treatment with TRAIL and wogonin

Background and purpose:

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptotic death in a variety of cancer cells without marked toxicity to most normal cells. We previously reported that wogonin, a potent anticancer agent from a Chinese herb, up-regulates p53 in prostate cancer cells. In this study, the effects of combinations of TRAIL and wogonin on a human prostate cancer cell line LNCaP, resistant to TRAIL, was evaluated for evidence of synergy in triggering apoptosis.

Experimental approach:

Western blot assay and the ‘comet’ assay were used to study the underlying mechanisms of cell death and search for any mechanisms of enhancement of TRAIL-induced apoptosis in the presence of wogonin.

Key results:

During combined treatment with wogonin and TRAIL, cytotoxicity, poly(ADP-ribose) polymerase cleavage and caspase activation were associated with up-regulation of p53 through DNA damage and reactive oxygen species (ROS) generation. N-acetylcysteine (NAC), an antioxidant, inhibited ROS generation and synergistic interaction between TRAIL and wogonin. Experimental results in human colon cancer HCT116 cells demonstrated that p53-dependent Puma up-regulation played an important role; deficiency in either p53 or Puma prevented wogonin-enhanced TRAIL-induced apoptosis.

Conclusions and implications:

The present studies suggest that wogonin enhances TRAIL-induced cytotoxicity through up-regulation of p53 and Puma, mediated by ROS.     

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.     

The effect of Echinacea purpurea on the pharmacokinetics of docetaxel

Aims

The herbal medicine Echinacea purpurea (E. purpurea) has been shown to induce cytochrome P450 3A4 (CYP3A4) both in vitro and in humans. This study explored whether E. purpurea affects the pharmacokinetics of the CYP3A4 substrate docetaxel in cancer patients.

Methods

Ten evaluable cancer patients received docetaxel (135 mg, 60 min IV infusion) before intake of a commercially available E. purpurea extract (20 oral drops three times daily) and 3 weeks later after a 14 day supplementation period with E. purpurea. In both cycles, pharmacokinetic parameters of docetaxel were determined.

Results

Before and after supplementation with E. purpurea, the mean area under the plasma concentration–time curve of docetaxel was 3278 ± 1086 and 3480 ± 1285 ng ml⁻¹ h, respectively. This result was statistically not significant. Nonsignificant alterations were also observed for the elimination half-life (from 30.8 ± 19.7 to 25.6 ± 5.9 h, P = 0.56) and maximum plasma concentration of docetaxel (from 2224 ± 609 to 2097 ± 925 ng ml⁻¹, P = 0.30).

Conclusions

The multiple treatment of E. purpurea did not significantly alter the pharmacokinetics of docetaxel in this study. The applied E. purpurea product at the recommended dose may be combined safely with docetaxel in cancer patients.