Resveratrol induces apoptosis of human chronic myelogenous leukemia cells in vitro through p38 and JNK-regulated H2AX phosphorylation

Aim:

The phosphorylation of histone H2AX, a novel tumor suppressor protein, is involved in regulation of cancer cell apoptosis. The aim of this study was to examine whether H2AX phosphorylation was required for resveratrol-induced apoptosis of human chronic myelogenous leukemia (CML) cells in vitro.

Methods:

K562 cells were tested. Cell apoptosis was analyzed using flow cytometry, and the phosphorylation of H2AX and other signaling proteins was examined with Western blotting. To analyze the signaling pathways, the cells were transfected with lentiviral vectors encoding H2AX-wt or specific siRNAs.

Results:

Treatment of K562 cells with resveratrol (20–100 μmol/L) induced apoptosis and phosphorylation of H2AX at Ser139 in time- and dose-dependent manners, but reduced phosphorylation of histone H3 at Ser10. Resveratrol treatment activated two MAPK family members p38 and JNK, and blocked the activation of another MAPK family member ERK. Pretreatment with the p38 inhibitor SB202190 or the JNK inhibitor SP600125 dose-dependently reduced resveratrol-induced phosphorylation of H2AX, which were also observed when the cells were transfected with p38- or JNK-specific siRNAs. Overexpression of H2AX in K562 cells markedly increased resveratrol-induced apoptosis, whereas overexpression of H2AX-139m (Ser139 was mutated to block phosphorylation) inhibited resveratrol-induced apoptosis. K562 cells transfected with H2AX-specific siRNAs were resistant to resveratrol-induced apoptosis.

Conclusion:

H2AX phosphorylation at Ser139 in human CML cells, which is regulated by p38 and JNK, is essential for resveratrol-induced apoptosis.     

Berberine induces apoptosis in SW620 human colonic carcinoma cells through generation of reactive oxygen species and activation of JNK/p38 MAPK and FasL

Berberine is the major constituent of Coptidis Rhizoma with multiple pharmacological activities, including anti-inflammation, promotion of apoptosis and anticancer potential effect. Mitogen-activated protein kinase (MAPK) and reactive oxygen species (ROS) may contribute to the causal relationship between tumorigenesis and pro-apoptotic function. Berberine is studied for the mechanism of its action in apoptotic pathway in human colonic carcinoma cell. Treatment of SW620 cells with 50 μM berberine resulted in activation of the caspase 3 and caspase 8, cleavage of poly ADP-ribose polymerase (PARP) and the release of cytochrome c; whereas, the expression of BID and anti-apoptosis factor c-IAP1, Bcl-2, and Bcl-_XL were decreased markedly. Berberine-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of JNK and p38 MAPK, as well as generation of the ROS. Furthermore, the induction of apoptosis was alleviated by inhibitors specific for JNK and p38. In addition, there was an increase in the cellular levels of phospho-c-Jun, FasL and t-BID in the berberine-induced apoptosis via the activation of JNK and p38 signaling modules. NAC administration, a scavenger of ROS, reversed berberine-induced apoptosis effects via inhibition of JNK, p38 and c-jun activation, and FasL and t-BID expression. These results leads us to speculate that berberine may play an apoptotic cascade in SW620 cells by activation of the JNK/p38 pathway and induction of ROS production, providing a new mechanism for berberine-induced cell death in human colon cancer cells.     

Crotoxin induces apoptosis and autophagy in human lung carcinoma cells in vitro via activation of the p38MAPK signaling pathway

Aim:

Crotoxin (CrTX) is the primary toxin in South American rattlesnake (Crotalus durissus terrificus) venom, and exhibits antitumor and other pharmacological actions in vivo and in vitro. Here, we investigated the molecular mechanisms of the antitumor action of CrTX in human lung carcinoma cells in vitro.

Methods:

Human lung squamous carcinoma SK-MES-1 cells were tested. The cytotoxicity of CrTX was evaluated in both MTT and colony formation assays. Cell cycle was investigated with flow cytometry. Cell apoptosis was studied with Hoechst 33258 and Annexin V-FITC staining. The levels of relevant proteins were analyzed using Western blot assays.

Results:

CrTX (25, 50, 100 μmol/L) inhibited the growth and colony formation of SK-MES-1 cells in dose- and time-dependent manners. CrTX increased the proportion of S phase cells and dose-dependently induced cell apoptosis, accompanied by down-regulating the expression of proliferating cell nuclear antigen (PCNA), and increasing the level of cleaved caspase-3. Furthermore, CrTX dose-dependently increased the expression of autophagy-related proteins LC3-II and beclin 1, and decreased the level of p62 in the cells. Moreover, CrTX (50 μmol/L) significantly increased p38MAPK phosphorylation in the cells. Pretreatment of the cells with SB203580, a specific inhibitor of p38MAPK, blocked the inhibition of CrTX on cell proliferation, as well as CrTX-induced apoptosis and cleaved caspase-3 expression.

Conclusion:

The p38MAPK signaling pathway mediates CrTX-induced apoptosis and autophagy of human lung carcinoma SK-MES-1 cells in vitro.     

Mig-2 attenuates cisplatin-induced apoptosis of human glioma cells in vitro through AKT/JNK and AKT/p38 signaling pathways

Aim:

Mig-2 (also known as Kindlin-2 and FERMT2) is an important regulator of integrin activation and cell-extracellular matrix adhesion, and involved in carcinogenesis and tumor progression. The aim of this study was to investigate the role of mig-2 in cisplatin-induced apoptosis of human glioma cells in vitro.

Methods:

The expression of mig-2 was modulated in human glioma H4, HS 683 and U-87 MG cells by transfection with a plasmid carrying mig-2 or mig-2 siRNA. Cisplatin-induced apoptosis was detected using Annexin V/PI staining and flow cytometry, as well as MTS analyses. The expression of apoptosis-related or signaling proteins was examined using Western blotting analysis. H4 cells were transfected with plasmids carrying mig-2 mutants to determine the functional domain of mig-2.

Results:

In the 3 glioma cell lines tested, overexpression of mig-2 significantly attenuated cisplatin-induced apoptosis, whereas knock-down of mig-2 potentiated the apoptosis. The mechanisms of action of mig-2 were further addressed in H4 cells: overexpression of mig-2 markedly reduced cleaved caspase-9, caspase-8, caspase-3 and PARP, as well as p-JNK and p-p38, and increased p-AKT in cisplatin-treated H4 cells, whereas mig-2 siRNA reversely changed these apoptosis-related and signaling proteins. Furthermore, pretreatment with JNK inhibitor SP600125 and p38 inhibitor SB203580, or with AKT inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 abolished the effects of mig-2 on cisplaxtin-induced apoptosis. In H4 cells, GFP-mig-2 F3 plasmid that contained only the F3 subdomain showed the same efficiency in attenuating cisplatin-induced apoptosis, as the mig-2 wild-type vector did, whereas GFP-mig-2 (1-541) plasmid that lacked the F3 subdomain was inactive.

Conclusion:

Mig-2 significantly attenuates the antitumor action of cisplatin against human glioma cells in vitro through AKT/JNK and AKT/p38 signaling pathways. The F3 subdomain of mig-2 is necessary and sufficient for this effect.     

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.     

酪酸梭菌对HT-29细胞水通道蛋白3表达的影响及JNK和p38信号通路对其调控作用

目的明确酪酸梭菌对人结肠腺癌细胞HT-29细胞水通道蛋白3(AQP3)m RNA和蛋白表达的影响以及c-Jun氨基末端激酶(JNK)和p38信号转导通路对其表达的调控作用。方法用酪酸梭菌上清液作用于HT-29细胞后,应用实时定量反转录聚合酶链反应(RT-PCR)和蛋白印迹方法检测AQP3 m RNA和蛋白表达水平。AQP3表达水平有差异后测定总JNK、p38以及磷酸化的JNK(P-JNK)和磷酸化的p38(P-p38)蛋白表达水平。后分别加用JNK抑制剂SP600125和p38抑制剂SB203580后测定AQP3蛋白表达量,观察JNK和p38对AQP3表达的调控作用。统计学处理采用单因素方差分析。结果实时定量RT-PCR和蛋白印迹法结果显示酪酸梭菌上清液不同浓度(1∶4和1∶8组)作用HT-29细胞12 h以及同一浓度1∶4作用不同时间(24、12、6 h组)的AQP3 m RNA和蛋白表达水平均较无酪酸梭菌上清液作用组明显上调(P均<0.05)。蛋白印迹法结果显示酪酸梭菌上清液组的Pp38/p38和P-JNK/JNK蛋白相对量较空白对照组表达水平明显上调(F=26.065和65.034,P<0.05)。蛋白印迹法结果显示加入p38抑制剂SB203580 10μg/ml组和20μg/ml的AQP3蛋白表达相对量为(0.7±0.4)和(0.6±0.6)、加入JNK抑制剂SP600125 10μg/ml组和20μg/ml的AQP3蛋白表达相对量为(0.7±0.4)和(0.5±0.4)均较未加入抑制剂组(1.1±0.7)AQP3蛋白表达水平明显下调(F=36.225,P<0.05)。结论酪酸梭菌上清液能够上调HT-29细胞的AQP3表达进而调节肠道的水代谢。JNK和p38信号转导通路可能参与调控AQP3的表达。     

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.     

Gambogic acid induces growth inhibition and differentiation via upregulation of p21waf1/cip1 expression in acute myeloid leukemia cells

Gambogic acid (GA) is the major active ingredient of gamboges, a brownish to orange resin product from Garcinia hanburyi tree in Southeast Asia. This compound exhibits anti-cancer effect on solid tumors. In this study, we investigated the effects of GA on the growth and differentiation of acute myeloid leukemia cells by growth-inhibition detection, morphological changes observation, nitroblue tetrazolium reduction, and the expression of the relative cell-surface differentiation markers. The results showed that GA could inhibit cell growth and promote differentiation in U937 and HL-60 cells. In addition, GA upregulated the expression of p21waf1/cip1 in the two cell lines. Finally, downregulating the p21waf1/cip1 expression with small interfering RNA partially blocked GA-induced cell growth inhibition and differentiation. These results of this study revealed that GA may be used as one of the investigational drugs for acute myeloid leukemia.     

PTPL1 suppresses lung cancer cell migration via inhibiting TGF-β1-induced activation of p38 MAPK and Smad 2/3 pathways and EMT

Epithelial-mesenchymal transition (EMT) enables dissemination of neoplastic cells and onset of distal metastasis of primary tumors. However, the regulatory mechanisms of EMT by microenvironmental factors such as transforming growth factor-β (TGF-β) remain largely unresolved. Protein tyrosine phosphatase L1 (PTPL1) is a non-receptor protein tyrosine phosphatase that plays a suppressive role in tumorigenesis of diverse tissues. In this study we investigated the role of PTPL1/PTPN13 in metastasis of lung cancer and the signaling pathways regulated by PTPL1 in terms of EMT of non-small cell lung cancer (NSCLC) cells. We showed that the expression of PTPL1 was significantly downregulated in cancerous tissues of 23 patients with NSCLC compared with adjacent normal tissues. PTPL1 expression was positively correlated with overall survival of NSCLC patients. Then we treated A549 cells in vitro with TGF-β1 (10 ng/mL) and assessed EMT. We found that knockdown of PTPL1 enhanced the migration and invasion capabilities of A549 cells, through enhancing TGF-β1-induced EMT. In nude mice bearing A549 cell xenografts, knockdown of PTPL1 significantly promoted homing of cells and formation of tumor loci in the lungs. We further revealed that PTPL1 suppressed TGF-β-induced EMT by counteracting the activation of canonical Smad2/3 and non-canonical p38 MAPK signaling pathways. Using immunoprecipitation assay we demonstrated that PTPL1 could bind to p38 MAPK, suggesting that p38 MAPK might be a direct substrate of PTPL1. In conclusion, these results unravel novel mechanisms underlying the regulation of TGF-β signaling pathway, and have implications for prognostic assessment and targeted therapy of metastatic lung cancer.     

Catalytic activity-independent pathway is involved in phospholipase A2-induced apoptotic death of human leukemia U937 cells via Ca-mediated p38 MAPK activation and mitochondrial depolarization

In view of the controversial role of catalytic activity on the cytotoxicity of phospholipase A₂ (PLA₂), the present study is conducted to explore whether PLA₂ induces apoptotic process of human leukemia U937 cells through catalytic activity-independent pathway. Modification of His-48 (according to the sequence alignment with porcine pancreatic PLA₂) with p-bromophenacyl bromide (BPB) caused over 99.9% drop in enzymatic activity Naja naja atra PLA₂. It was found that BPB–PLA₂-induced apoptotic death of U937 cells was associated with mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Upon exposure to BPB–PLA₂, elevation of intracellular Ca²⁺ levels and p38 MAPK activation were observed in U937 cells. Pretreatment with BAPTA-AM (Ca²⁺ chelator) and nifedipine (L-type Ca²⁺ channel blocker) abrogated Ca²⁺ increase and p38 MAPK activation, and rescued viability of BPB–PLA₂-treated U937 cells. BPB–PLA₂-induced dissipation of mitochondrial membrane potential and down-regulation of Bcl-2 were suppressed by SB202190 (p38MAPK inhibitor). Although PLA₂ mutants in which His-48 and Asp-49 were substituted by Ala and Lys, respectively, did not display detectable PLA₂ activity, they induced death of U937 cells. The signaling pathway of PLA₂ mutants in inducing cell death was indistinguishable from that of BPB–PLA₂. Taken together, our data indicate that catalytic activity-independent pathway is involved in PLA₂-induced apoptotic death of human leukemia U937 cells via mitochondria-mediated death pathway triggering by Ca²⁺-mediated p38 MAPK activation.     

2,3′,4,4′,5′-Pentamethoxy-trans-stilbene, a resveratrol derivative, is a potent inducer of apoptosis in colon cancer cells via targeting microtubules

Resveratrol, a naturally occurring polyphenolic antioxidant, is a compound holding promise for cancer chemoprevention. Previous studies suggest that 2,3′,4,5′-tetramethoxy-trans-stilbene (TMS) and 3,4,4′,5,-tetramethoxy-trans-stilbene (MR-4), both of which are derivatives of resveratrol, are potent apoptosis-inducing agents with clinical potential. In this study, we chemically synthesized 2,3′,4,4′,5′-pentamethoxy-trans-stilbene (PMS), the hybrid molecule of TMS and MR-4, and determined its effects on colon cancer growth. When compared with its parent compounds, PMS displayed more potent in vitro anti-mitogenic effect on colon cancer cells (Caco-2, HT-29 and SW1116). Moreover, PMS inhibited tumor growth in vivo in a colon cancer xenograft model. In this connection, PMS strongly induced apoptosis in HT-29 cells as evidenced by increased PARP cleavage, DNA fragmentation, and accumulation of sub-G₁ population. Further mechanistic analysis revealed that PMS enhanced the polymerization of microtubules, which was followed by G₂/M mitotic arrest and caspase-dependent apoptosis. The activation of caspases-3, -7, -8, and -9 was involved in PMS-induced apoptosis with concomitant down-regulation of the pro-survival PI3K/Akt signaling. Collectively, these data suggest that PMS is a potent inducer of apoptosis via targeting microtubules and may merit investigation as a potential chemoprophylactic and therapeutic agent for colon cancer.     

Increased apoptotic efficacy of lonidamine plus arsenic trioxide combination in human leukemia cells. Reactive oxygen species generation and defensive protein kinase (MEK/ERK, Akt/mTOR) modulation

Lonidamine is a safe, clinically useful anti-tumor drug, but its efficacy is generally low when used in monotherapy. We here demonstrate that lonidamine efficaciously cooperates with the anti-leukemic agent arsenic trioxide (ATO, Trisenox™) to induce apoptosis in HL-60 and other human leukemia cell lines, with low toxicity in non-tumor peripheral blood lymphocytes. Apoptosis induction by lonidamine/ATO involves mitochondrial dysfunction, as indicated by early mitochondrial permeability transition pore opening and late mitochondrial transmembrane potential dissipation, as well as activation of the intrinsic apoptotic pathway, as indicated by Bcl-X_L and Mcl-1 down-regulation, Bax translocation to mitochondria, cytochrome c and Omi/HtrA2 release to the cytosol, XIAP down-regulation, and caspase-9 and -3 cleavage/activation, with secondary (Bcl-2-inhibitable) activation of the caspase-8/Bid axis. Lonidamine stimulates reactive oxygen species production, and lonidamine/ATO toxicity is attenuated by antioxidants. Lonidamine/ATO stimulates JNK phosphorylation/activation, and apoptosis is attenuated by the JNK inhibitor SP600125. In addition, lonidamine elicits ERK and Akt/mTOR pathway activation, as indicated by increased ERK, Akt, p70S6K and rpS6 phosphorylation, and these effects are reduced by co-treatment with ATO. Importantly, co-treatment with MEK/ERK inhibitor (U0126) and PI3K/Akt (LY294002) or mTOR (rapamycin) inhibitors, instead of ATO, also potentiates lonidamine-provoked apoptosis. These results indicate that: (i) lonidamine efficacy is restrained by drug-provoked activation of MEK/ERK and Akt/mTOR defensive pathways, which therefore represent potential therapeutic targets. (ii) Co-treatment with ATO efficaciously potentiates lonidamine toxicity via defensive pathway inhibition and JNK activation. And (iii) conversely, the pro-oxidant action of lonidamine potentiates the apoptotic efficacy of ATO as an anti-leukemic agent.