Annosquacin B induces mitochondrial apoptosis in multidrug resistant human breast cancer cell line MCF-7/ADR through selectively modulating MAPKs pathways

Context: Multidrug resistance (MDR) is a major obstacle to efficient therapy of cancers. It is a prime concern for researchers to find compounds with anti-proliferative activity on MDR cell lines. In recent years, annonaceous acetogenins (ACGs) were reported to have anti-proliferative activity. However, the underlying mechanisms are still unknown.

Objective: This study determines the mechanisms of anti-proliferative activity induced by Annosquacin B (AB) against MCF-7/ADR cells.

Material and methods: The cytotoxicity of AB at varying concentrations (0.64, 1.6, 4, 10, 25, 62.5, 156.25?μM) on MCF-7/ADR cells was assessed using the MTT assay. Annexin V-FITC/propidium iodide staining and Acrinidine orange and ethidium bromide (AO/EB) staining were employed to investigate whether AB (14, 7, 3.5?μM) could induce apoptosis in MCF-7/ADR cells. Levels of caspase-3 and caspase-9, Bax, Bcl-2 and MAPKs kinases were evaluated by western blot assay following treatment with various concentrations of AB (3.5, 7, 14?μM) at different time points (0, 0.5, 1, 2, 4, 8, 12?h).

Results and conclusion: MTT assay showed that AB significantly decreased cell viability on MCF-7/ADR (IC₅₀ of 14.69?μM). AB-induced apoptosis in MCF-7/ADR cells through mitochondrial apoptosis pathways. It induced typical apoptosis by morphologic changes; elevate levels of caspase-3, caspase-9 as well as the ratio of Bax/Bcl-2. In addition, AB increased the expression of p-p38 MAPK and decreased the expression of p-JNK, while whether ERK1/2 had an effect on the MCF-7/ADR apoptosis remains to be determined.     

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.     

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.     

Geraniin induces apoptosis of human breast cancer cells MCF-7 via ROS-mediated stimulation of p38 MAPK

Geraniin, a typical ellagitannin isolated from Phyllanthus urinaria Linn, has been found to possess a range of bioactive properties. In the present study, we found that Geraniin showed potent anti-proliferative effects on human breast cancer MCF-7 cells. The IC₅₀ values were 9.94, 17.98 and 42.32?µM after 72-, 48- and 24-h treatment, respectively. Meanwhile, Geraniin could remarkably disrupt mitochondrial membrane potential and arrest S phase cell cycle. Western-blot analysis showed that Geraniin induced phosphorylation of the anti-apoptotic Bcl-2, and the cleavage of poly (ADP-ribose) polymerase (PARP) and caspase-3 in MCF-7 cells. Moreover, Geraniin treatment activated p38 mitogen-activated protein kinase (p38 MAPK) and the effect was blunted in MCF-7 cells with the treatment of a specific p38 inhibitor SB203580. Geraniin could generate intracellular reactive oxygen species (ROS), activate p38 MAPK then induce the apoptosis in MCF-7 cells, such phenomena was abrogated by pretreatment with N-acetyl-l-cysteine. In general, these results support the conclusion that Geraniin-induced apoptosis is mediated via ROS-mediated stimulation of p38 MAPK signaling.     

S-allylcysteine,a garlic derivative,suppresses proliferation and induces apoptosis in human ovarian cancer cells in vitro

Aim： To investigate the effects of S-allylcysteine （SAC）, a water-soluble garlic derivative, on human ovarian cancer cells in vitro. Methods： Human epithelial ovarian cancer cell line A2780 was tested. Cell proliferation was examined with CCK-8 and colony formation assays. Cell cycle was analyzed with flow cytometry. Cell apoptosis was studied using Hoeohst 33258 staining and Annexin V/PI staining with flow cytometry. The migration and invasion of A2780 cells were examined with transwell and wound healing assays. The expression of relevant proteins was detected with Western blot assays. Results： SAC （1-100 mmol/L） inhibited the proliferation of A2780 cells in dose- and time-dependent manners （the ICsovalue was approximately 25 mmoVL at 48 h, and less than 6.25 mmol/L at 96 h）. Furthermore, SAC dose-dependently inhibited the colony formation of A2780 cells. Treatment of A2780 cells with SAC resulted in G/S phase arrest and induced apoptosis, accompanied by decreased expression of pro-caspase-3, Parp-1 and Bcl-2, and increased expression of active caspase-3 and Bax. SAC treatment significantly reduced the migration of A2780 cells, and markedly decreased the protein expression of Wnt5a, p-AKT and c-Jun, which were the key proteins involved in proliferation and metastasis. Conclusion： SAC suppresses proliferation and induces apoptosis in A2780 ovarian cancer cells in vitro.     

海绵来源的smenospongine诱导乳腺癌MCF7细胞凋亡机制研究

目的 研究海绵Spongia pertusa Esper来源的smenospongine（Sme）诱导乳腺癌细胞MCF7凋亡的作用机制。方法 使用CCK-8法检测Sme对MCF7细胞活力的影响;DAPI染色检测凋亡细胞的细胞核形态;使用流式细胞术检测细胞凋亡率和线粒体膜电位;Western blotting检测Sme对Bax、Bcl2、细胞色素C（cytochorome C）、p-p38和p38蛋白水平表达的影响。结果 CCK-8法检测结果表明,Sme抑制MCF7增殖,IC₅₀值为（16.46 ±0.88）μmol/L;DAPI染色结果和Annexin V-FITC/PI染色结果显示Sme诱导细胞凋亡。随着加药浓度的增加,细胞凋亡率从4.18%逐渐升至21.49%;流式细胞仪检测结果表明Sme引发MCF7细胞内线粒体膜电位下降;Western blotting结果显示Sme激活了内源性凋亡途径和p38丝裂源活化蛋白激酶（MAPK）信号通路。结论 Sme可能通过激活p38 MAPK通路,诱导细胞内源性凋亡发挥抗乳腺癌作用。     

Platycodin D induces apoptosis and triggers ERK- and JNK-mediated autophagy in human hepatocellular carcinoma BEL-7402 cells

Aim:

Platycodin D, the main saponin isolated from Chinese herb Platycodonis Radix, exhibits anticancer activities against various cancer cell lines. Here we evaluated its anticancer action against human hepatocellular carcinoma cells in vitro and in vivo, and elucidated the relationship between platycodin D-induced apoptosis and autophagy.

Methods:

The viability of human hepatocellular carcinoma BEL-7402 cells was evaluated with MTT assay, and the apoptosis was examined using Annexin V/PI and Hoechst 33342 staining assays. Monodansylcadaverine (MDC) staining was used to label autophagic vacuoles. The proteins were detected using Western blot analysis. For studying its anticancer action in vivo, platycodin D (5 and 10 mg· kg⁻¹·d⁻¹) was intraperitoneally injected to BEL-7402-bearing mice for 21 days.

Results:

Platycodin D (5–40 μmol/L) inhibited the cell proliferation in vitro with IC50 values of 37.70±3.99, 24.30±2.30 and 19.70±2.36 μmol/L at 24, 48 and 72 h, respectively. Platycodin D (5–20 μmol/L) dose-dependently increased BEL-7402 cell apoptosis, increased the Bax/Bcl-2 ratio and the levels of cleaved PARP and cleaved caspase-3, and decreased the level of Bcl-2. Furthermore, platycodin D (5–20 μmol/L) induced autophagy in BEL-7402 cells, as evidenced by formation of cytoplasmic vacuoles, increased amounts of LC3-II, and increased numbers of MDC-positive cells. Pretreatment with the autophagy inhibitor chloroquine (5 μmol/L) or BAF (50 nmol/L) significantly enhanced platycodin D-induced proliferation inhibition and apoptosis. Moreover, platycodin D (20 μmol/L) activated the ERK and JNK pathways in BEL-7402 cells, and simultaneous blockage of the two pathways effectively suppressed platycodin D-induced autophagy and enhanced platycodin D-induced apoptosis. In BEL-7402-bearing mice, platycodin D (10 mg·kg⁻¹•d⁻¹) significantly reduced relative tumor volume with decreased body weight.

Conclusion:

Platycodin D not only inhibits the proliferation of BEL-7402 cells but also suppresses BEL-7402 xenograft tumor growth. Platycodin D-induced cell proliferation inhibition and apoptosis are amplified by co-treatment with autophagy inhibitors     

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.     

Synergistic suppression of human breast cancer cells by combination of plumbagin and zoledronic acid In vitro

Aim:

Zoledronic acid (ZA), a bisphosphonate, is currently used in combination with chemotherapeutic agents to suppress breast cancer cell proliferation or breast cancer-induced osteolysis. The aim of this study was to investigate the effects of ZA combined with a natural anticancer compound plumbagin (PL) against human breast cancer cells in vitro.

Methods:

Human breast cancer MDA-MB-231SArfp cells were treated with ZA, PL or a combination of ZA and PL. The cell growth, apoptosis and migration were evaluated using CCK-8 assay, flow cytometry and transwell assay, respectively. The expression of apoptosis-related proteins was measured using real-time PCR and Western blotting. Synergism was evaluated using Compusyn software, and the combination index (CI) and drug reduction index (DRI) values were determined.

Results:

PL or ZA alone caused mild cytotoxicity (the IC50 value at 24 h was 12.18 and above 100 μmol/L, respectively). However, the combination of ZA and PL caused a synergistic cytotoxicity (CI=0.26). The DRI values also showed a synergistic effect between PL and ZA, with actual values of 5.52 and 3.59, respectively. Furthermore, PL and ZA synergistically induced apoptosis and inhibited migration of the breast cancer cells. Moreover, the combination of ZA and PL decreased the expression of Notch-1, cleaved PARP, Bcl-2 and Bcl-xl, and increased the expression of cleaved caspase-3, CDKN1A and ID1. When the breast cancer cells were transfected with specific siRNA against Notch-1, the combination of ZA and PL markedly increased the expression of Bcl-2.

Conclusion:

Combination of ZA and PL synergistically suppresses human breast cancer MDA-MB-231SArfp cells in vitro. PL can inhibit ZA-induced activation of the Notch-1 signaling pathway and subsequently reduce the expression of Bcl-2, thus potentiating cancer cell apoptosis.     

Bufalin induces apoptosis via mitochondrial ROS-mediated caspase-3 activation in HCT-116 and SW620 human colon cancer cells

Objective: Bufalin has been reported to kill various types of cancer including human colorectal cancer. Our previous study demonstrated that bufalin induced cell death via autophagy in HT-29 and Caco-2 colon cancer cells, but the action of bufalin remains unclear. This study was conducted to investigate the role of bufalin in other colon cancer HCT-116 and SW620 cells as well as its potential mechanism.

Methods: The effect of bufalin in HCT-116 and SW620 colon cancer cells was detected by assessing cell viability and cell death. Apoptotic cells were analyzed by Western blot and trypan blue dye exclusion assay. Mitochondrial ROS production was analyzed by flow cytometry after DCFDA and DHR-123 staining. The potential mechanism was investigated via pharmacological inhibitors.

Results: Bufalin had high potency against HCT-116 and SW620 cells with IC50 values of 12.823?±?1.792?nM and 26.303?±?2.498?nM in HCT-116 and SW620 cells, respectively. Bufalin decreased cell viability, increased cell death as well as caspase-3 downstream target (cleaved PARP) accumulation, and these actions were significantly blocked by pan-caspase inhibitor zVAD-FMK. Mechanistically, ROS production, but neither the NAD(P)H oxidase, AMPK, ERK nor p38, is responsible for bufalin-induced apoptotic cell death. Moreover, bufalin-induced ROS generation is derived from mitochondria.

Conclusion: Bufalin significantly induces apoptosis in HCT-116 and SW620 colon cancer cells via mitochondrial ROS-mediated caspase-3 activation. We believe that our novel findings will greatly alter our current understanding on the anti-cancer mechanism of bufalin in colon cancer cells and will pave the way for further exploiting the clinical application.     

Novel ferrocenyl derivatives exert anti-cancer effect in human lung cancer cells in vitro via inducing G1-phase arrest and senescence

Aim:

To investigate the effects of 7 novel 1-ferrocenyl-2-(5-phenyl-1H-1,2,4-triazol-3-ylthio) ethanone derivatives on human lung cancer cells in vitro and to determine the mechanisms of action.

Methods:

A549 human lung cancer cells were examined. Cell viability was analyzed with MTT assay. Cell apoptosis and senescence were examined using Hoechst 33258 and senescence-associated-β-galactosidase (SA-β-gal) staining, respectively. LDH release was measured using a detection kit. Cell cycle was analyzed using a flow cytometer. Intracellular ROS level was measured with the 2′,7′-dichlorodihydrofluorescein probe. Phosphorylation of p38 was determined using Western blot.

Results:

Compounds 5b, 5d, and 5e (40 and 80 μmol/L) caused significant decrease of A549 cell viability, while other 4 compounds had no effect on the cells. Compounds 5b, 5d, and 5e (80 μmol/L) induced G₁-phase arrest (increased the G₁ population by 22.6%, 24.23%, and 26.53%, respectively), and markedly increased SA-β-gal-positive cells. However, the compounds did not cause nuclear DNA fragmentation and chromatin condensation in A549 cells. Nor did they affect the release of LDH from the cells. The compounds significantly elevated the intracellular ROS level, decreased the mitochondrial membrane potential, and increased p38 phosphorylation in the cells. In the presence of the antioxidant and free radical scavenger N-acetyl-L-cysteine (10 mmol/L), above effects of compounds 5b, 5d, and 5e were abolished.

Conclusion:

The compounds 5b, 5d, and 5e cause neither apoptosis nor necrosis of A549 cells, but exert anti-cancer effect via inducing G₁-phase arrest and senescence through ROS/p38 MAP-kinase pathway.     

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.     

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.     

Involvement of reactive oxygen species/c-Jun NH(2)-terminal kinase pathway in kotomolide A induces apoptosis in human breast cancer cells

The anticancer effects of kotomolide A (KTA), a new butanolide constituent isolated from the leaves of Cinnamomum kotoense (Lauraceae), on the two human breast cancer cell lines MCF-7 and MDA-MB-231, were first investigated in our study. KTA exhibited selectively antiproliferative effects in cancer cell lines without showing any toxicity in normal mammary epithelial cells. Treatment of cancer cells with KTA to trigger G2/M phase arrest was associated with increased p21/WAF1 levels and reduced amounts of cyclin A, cyclin B1, cdc2 and cdc25C. KTA induced cancer cell death treatment by triggering mitochondrial and death receptor 5 (DR5) apoptotic pathways, but did not act on the Fas receptor. Exposure of MCF-7 and MDA-MB-231 cells to KTA resulted in cellular glutathione reduction and ROS generation, accompanied by JNK activation and apoptosis. Both antioxidants, NAC and catalase, significantly decreased apoptosis by inhibiting the phosphorylation of JNK and subsequently triggering DR5 cell death pathways. The reduction of JNK expression by siRNA decreased KTA-mediated Bim cleavage, DR5 upregulation and apoptosis. Furthermore, daily KTA i.p. injections in nude mice with MDA-MB-231 s.c. tumors resulted in a 50% decrease of mean tumor volume, compared with vehicle-treated controls. Taken together, the data show that cell death of breast cancer cells in response to KTA is dependent upon ROS generation and JNK activation, triggering intrinsic and extrinsic apoptotic pathways. The ROS/JNK pathway could be a useful target for novel approaches in breast cancer chemotherapy.     

Capsaicin induces apoptosis in SCC-4 human tongue cancer cells through mitochondria-dependent and -independent pathways

Although there have been advances in the fields of surgery, radiotherapy, and chemotherapy of tongue cancer, the cure rates are still not substantially satisfactory. Capsaicin (trans‐8‐methyl‐N‐vanillyl‐6‐nonenamide) is the major pungent ingredient of hot chili pepper and has been reported to have an antitumor effect on many human cancer cell types. The molecular mechanisms of the antitumor effect of capsaicin are not yet completely understood. Herein, we investigated whether capsaicin induces apoptosis in human tongue cancer cells. Capsaicin decreased the percentage of viable cells in a dose‐dependent manner in human tongue cancer SCC‐4 cells. In addition, capsaicin produced DNA fragmentation, decreased the DNA contents (sub‐G1 phase), and induced G0/G1 phase arrest in SCC‐4 cells. We demonstrated that capsaicin‐induced apoptosis is associated with an increase in reactive oxygen species and Ca²⁺ generations and a disruption of the mitochondrial transmenbrane potential (ΔΨ_m). Treatment with capsaicin induced a dramatic increase in caspase‐3 and ‐9 activities, as assessed by flow cytometric methods. A possible mechanism of capsaicin‐induced apoptosis is involved in the activation of caspase‐3 (one of the apoptosis‐executing enzyme). Confocal laser microscope examination also showed that capsaicin induced the releases of AIF, ATF‐4, and GADD153 from mitochondria of SCC‐4 cells. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

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.     

YC-1 induces apoptosis of human renal carcinoma A498 cells in vitro and in vivo through activation of the JNK pathway

Background and purpose:

The aim of this study was to elucidate the mechanism of YC-1{3-(5′-hydroxy methyl-2′-furyl)-1-benzylindazole}-induced human renal carcinoma cells apoptosis and to evaluate the potency of YC-1 in models of tumour growth in mice.

Experimental approach:

YC-1-mediated apoptosis was assessed by analysis of MTT, SRB, DAPI staining and flow cytometry analysis. Knockdown of JNK protein was achieved by transient transfection using siRNA. The mechanisms of action of YC-1 on different signalling pathways involved were studied using western blot. Fas clustering was analysed by confocal microscopy and in vivo efficacy was examined in a A498 xenograft model.

Key results:

YC-1 displayed cytotoxicity in renal carcinoma cells at 10⁻⁷–10⁻⁸ M. Increased condensation of chromatin was observed and an increase in the cell population in subG1 phase. Moreover, YC-1 triggered mitochondria-mediated and caspase-dependent pathways. YC-1 significantly induced Fas ligand expression, but did not modify either the protein levels of death receptors or ligands. In addition, Fas clustering in cells responsive to YC-1 was observed, suggesting involvement of a Fas-mediated pathway. Furthermore, YC-1 markedly induced phosphorylation of JNK and a JNK inhibitor, SP600125, and siRNA JNK1/2 significantly reversed YC-1-induced cytotoxicity and protein expression. We suggest that YC-1 induced JNK phosphorylation, the upregulation of FasL and Fas receptor clustering to promote the activation of caspases 8 and 3, resulting in apoptosis. Finally, we demonstrated the antitumour effect of YC-1 in vivo.

Conclusions and implications:

These data suggest that YC-1 is a good candidate for development as an anticancer drug.     

天花粉对人乳腺癌MCF-7细胞凋亡及相关基因表达的影响

目的研究天花粉对人乳腺癌MCF-7细胞凋亡及相关基因表达的影响。方法取对数生长期人乳腺癌MCF-7细胞以30,60,90,120μg·m L^-1的天花粉蛋白处理,作为实验组,对照组用等量的0.9%Na Cl处理,继续培养24,48,72h。观察并比较各组细胞形态学及细胞核凋亡形态学变化,以噻唑蓝(MTT)比色法检测各组人乳腺癌MCF-7细胞增殖抑制情况,分光光度法检测天花粉蛋白对人乳腺癌MCF-7细胞胱天蛋白酶(caspase)-3和caspase-8表达的影响。结果实验组人乳腺癌MCF-7细胞呈现明显的细胞凋亡及核凋亡现象,且随天花粉蛋白质量浓度的增加,其细胞核凋亡现象越明显;干预24 h后,30,60,90,120μg·mL^-1实验组增殖抑制率分别为(1.61±0.94)%,(2.81±1.01)%,(7.05±1.03)%和(9.59±1.12)%;干预48 h后增殖抑制率分别为(2.13±1.01)%,(6.14±1.12)%,(9.05±1.09)%和(19.60±1.15)%;干预72 h后增殖抑制率分别为(3.28±1.07)%,(7.18±1.51)%,(18.39±1.81)%和(29.59±1.63)%。实验组人乳腺癌MCF-7细胞增殖抑制率随作用时间的延长及天花粉蛋白质量浓度的增大而逐渐升高(均P<0.01);90μg·m L^-1实验组24,48,72 h caspase-3分别为1.49±0.15,2.36±0.25,2.15±0.23;caspase-8分别为1.94±0.26,1.56±0.19,1.39±0.20。对照组人乳腺癌MCF-7细胞caspase-3及caspase-8随作用时间的延长无显著变化(均P>0.05),而90μg·mL^-1实验组caspase-3先升高后降低(P<0.05或P<0.01),以48 h时最高;caspase-8则逐渐降低(P<0.01);且各时间点90μg·m L^-1实验组caspase-3及caspase-8均显著高于对照组(均P<0.01)。结论天花粉蛋白可有效促进人乳腺癌MCF-7细胞凋亡,抑制其增殖,且增殖抑制作用呈时间-剂量依赖性,其作用机制可能与上调人乳腺癌MCF-7细胞caspase-3及caspase-8表达相关。