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.     

In vitro and in vivo antitumour activities of puerarin 6″-O-xyloside on human lung carcinoma A549 cell line via the induction of the mitochondria-mediated apoptosis pathway

Context Pueraria lobata (Leguminoseae) shows cytotoxic effects against cancer cells; however, its active components remain unclear.

Objective This study investigated the antitumour activity of puerarin 6″-O-xyloside (POS) on the human lung carcinoma A549 cell line.

Materials and methods The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the cytotoxicity of POS (at 10, 20 and 40?μM) in vitro, and xenograft nude mice were established to evaluate the antitumour effect of POS (at 40?mg/kg/d) in vivo by 15 days intraperitoneal injection (ip). To explore its mechanism of action, flow cytometry was performed to determine the pro-apoptotic effect of POS (at 10, 20 and 40?μM). Subsequently, the expression of caspase-3, caspase-7, caspase-9, Bcl-2 and Bax in A549 cells were determined.

Results POS showed significant cytotoxicity toward A549 cells (p?<?0.05) by inducing apoptosis. Treatment with POS significantly upregulated the levels of caspase-3 (p?<?0.01), caspase-7 (p?<?0.01), caspase-9 (p?<?0.01) and Bax (p?<?0.01) in A549 cells, and Bcl-2 was downregulated (p?<?0.01). Additionally, the in vivo animal study showed that POS significantly inhibited tumour growth in A549 cells (p?<?0.01).

Conclusion Our study demonstrated the POS has significant antitumour activities. The mechanisms are related to increased levels of caspase-3, caspase-7, caspase-9 and Bax, and reduced levels Bcl-2.     

Neuroprotective effects of dimerumic acid and deferricoprogen from Monascus purpureus NTU 568-fermented rice against 6-hydroxydopamine-induced oxidative stress and apoptosis in differentiated pheochromocytoma PC-12 cells

Context Oxidative stress plays a key role in neurodegenerative disorders, including Parkinson’s disease (PD). Rice fermented with Monascus purpureus Went (Monascaceae) NTU 568 (red mould rice) was found to contain antioxidants, including dimerumic acid (DMA) and deferricoprogen (DFC).

Objective The effects of DMA and DFC on 6-hydroxydopamine (6-OHDA)-induced cytotoxicity and potential protective mechanisms in differentiated PC-12 pheochromocytoma cells were investigated.

Materials and methods DMA (0–60?μM) or DFC (0–10?μM) was co-treated with 6-OHDA (200?μM, 24?h exposure) in differentiated PC-12 cells. Cell viability and intercellular reactive oxygen species (ROS) were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) assays, respectively. Cell apoptosis was determined by DNA fragmentation analysis and propidium iodide staining by flow cytometry. Western blot analysis was used to measure the levels of cell protein expression.

Results DMA and DFC significantly increased cell viability to 72% and 81% in 6-OHDA-induced differentiated PC-12 cell cultures, respectively. Furthermore, DMA and DFC reduced 6-OHDA-induced formation of extracellular and intercellular ROS by 25% and 20%, respectively, and decreased NADPH oxidase-2 expression in differentiated PC-12 cells. DMA and DFC inhibited 6-OHDA-induced apoptosis and decreased activation of caspase-3 via regulation of Bcl-2-associated X protein (Bax) and Bcl-2 protein expression in differentiated PC-12 cells.

Conclusion DMA and DFC may protect against 6-OHDA toxicity by inhibiting ROS formation and apoptosis. These results showed that the metabolites from M. purpureus NTU 568 fermentation were potential therapeutic agents for PD induced by oxidative damage and should be encouraged for further research.     

Anticancer effect of 2,7-dihydroxy-3-methylanthraquinone on human gastric cancer SGC-7901 cells in vitro and in vivo

Context: 2,7-Dihydroxy-3-methylanthraquinone (DDMN) is reported to have a remarkable anticancer activity against gastric cancer SGC-7901 cells.

Objective: The objective of this study is to study the anticancer effect and mechanism of DDMN on SGC-7901 cells.

Materials and methods: The MTT assay was used to determine the effect of DDMN on cell viability of SGC-7901 cells, and the cytotoxic effect was evaluated by the IC₅₀ value. After treatment with different doses of DDMN (10, 20, and 40?μM) for 48?h, flow cytometry was used to investigate the apoptosis of SGC-7901 cells induced by DDMN. Further, western blotting was performed to study anticancer mechanism by assaying apoptosis-related proteins containing Mcl-1, Bcl-xl, Bcl-2, Bax, Bak, Bad, cytochrome c, caspase-3, and caspase-9. Finally, xenograft assay was used to further evaluate the effect of DDMN on SGC-7901 cells by determining body weight of nude mice, tumor volumes, and apoptosis-related proteins.

Results: These results suggest that DDMN can significantly inhibit (IC₅₀ value?=?20.92?μM) the proliferation of SGC-7901 cells and induce apoptosis of SGC-7901 cells demonstrated by flow cytometry analysis. Additionally, the results of western blotting indicated that DDMN can suppress the expression of anti-apoptotic proteins Bcl-xl and Bcl-2, increase the expression of pro-apoptotic proteins Bax, Bad (40?μM), caspase-3 and caspase-9, and evidently promote the release of cytochrome c from the mitochondria to the cytoplasm. The xenograft assay further confirmed that DDMN had significant anticancer effects on SGC-7901 cells.

Conclusion: DDMN had significant anticancer effect on SGC-7901 cells in vitro and in vivo related to mitochondria-mediated apoptosis.     

Corosolic acid analogue,a natural triterpenoid saponin,induces apoptosis on human hepatocarcinoma cells through mitochondrial pathway in vitro

Context 2a,-3a,-24-Trihydroxyurs-12-en-28-oic acid (TEO, a corosolic acid analogue) is a triterpenoid saponin isolated from Actinidia valvata Dunn (Actinidiaceae), a well-known traditional Chinese medicine.

Objective This study investigated the anti-proliferation and inducing apoptosis effects of TEO in three human hepatocellular carcinoma (HCC) cell lines.

Materials and methods Cytotoxic activity of TEO was determined by the MTT assay at various concentrations from 2.5 to 40?μg/mL in BEL-7402, BEL-7404 and SMMC-7721 cell lines. Cell morphology was assessed by acridine orange/ethidium bromide and 4′-6-diamidino-2-phenylindole dihydrochloride staining and fluorescence microscopy. Cell-cycle distribution and DNA damage were determined by flow cytometry and comet assay. Mitochondrial dysfunction was assessed by JC-1 staining and transmission electron microscopy. Apoptosis changes were explored by Western blot, TNF-α and caspase-3, -8, -9 assays.

Results TEO exhibited inhibition effects on BEL-7402, BEL-7404 and SMMC-7721 cells treated for 24?h, the IC₅₀ values were 34.6, 30.8 and 30.5?μg/mL, respectively. TEO (40?μg/mL)-treated three cell lines increased by more than 21% in the G1 phase and presented the morphological change and DNA damage. TEO also declined the mitochondrial membrane potential and altered mitochondrial ultra-structure. Furthermore, caspase-3, caspase-8, caspase-9 and TNF-α were also activated. Mechanism investigation showed that TEO could decrease anti-apoptotic Bcl-2 protein expression, increase proapoptotic Bax and Bid proteins expressions and increase Bax/Bcl-2 ratio.

Conclusion Our results demonstrate for the first time that TEO inhibited growth of HCC cell lines and induced G1 phase arrest. Moreover, proapoptotic effects of TEO were mediated through the activation of TNF-α, caspases and mitochondrial pathway.     

The antitumour effects of eudesmin on lung cancer by inducing apoptosis via mitochondria-mediated pathway in the tumour cells

Context: Limonoids possess broad range of biological activities, including antitumour, antimicrobial and antioxidant activities, etc. Eudesmin (EDN) is a type of limonoid which also possesses various activities. However, there is no report on the antitumour lung cancer (LC) activities of this compound.

Objective: The present study investigates the antitumour effects of EDN and its potential molecular mechanisms.

Materials and methods: The in vitro antitumour effects of EDN on LC A549 cells were evaluated by using MTT assay. The in vivo antitumour effects were investigated on a xenograft athymic nude mouse model. The mice were administered orally with EDN (10, 20 and 40?mg/kg) once daily for 28 days. Effects of EDN on apoptosis-related or signalling proteins (Bcl-2, Bax, caspase-3, caspase-9, P53, Akt and JNK) were assayed by western blot analysis.

Results: EDN showed significant inhibitory effects on the growth of LC A549 cells in vitro with the half maximal inhibitory concentration (IC₅₀) of 18.3?μM. By treating with EDN (10, 20 and 40?μM), expression of caspase-3, caspase-9, Bax, P53 and phosphorylated JNK in A549 cells were significantly upregulated, whereas expression of Bcl-2 and Akt phosphorylation were significantly down-regulated. Interestingly, EDN-induced apoptosis could be attenuated by JNK inhibitor. In addition, in vivo experiments also indicated EDN (10, 20 and 40?mg/kg) had significant antitumour effects (p?Conclusions: Overall, the results indicated that EDN possesses significant antitumour effects on LC and the possible mechanism might be related to induction of mitochondria-mediated apoptosis.     

Quercetin stimulates mitochondrial apoptosis dependent on activation of endoplasmic reticulum stress in hepatic stellate cells

Context: Activation of hepatic stellate cells (HSCs) is a hallmark of liver fibrosis. Quercetin has benefits for liver fibrosis, but the mechanisms are unknown.

Objective: We investigated the quercetin effect on HSC survival and the role of endoplasmic reticulum stress (ERS).

Materials and methods: Rat HSCs and LO2 hepatocytes were treated with quercetin (0.5–120?μM) for 24?h. Quercetin (10–40?μM) effects on apoptosis for 24?h were analyzed by flow cytometry and TUNEL staining. Quercetin (10–40?μM) effects on the expression of Bcl-2, caspase-9, caspase-3, PARP-1, PERK, IRE1, ATF6, calnexin and CHOP for 24?h were analyzed by Western blot. Quercetin (10–40?μM) effects on mRNA expression of calnexin and CHOP for 24?h were analyzed by Real-time PCR.

Results: Quercetin at concentrations greater than 20?μM significantly inhibited HSC proliferation (IC₅₀ 27.2?μM), but did not affect hepatocyte growth until 80?μM (IC₅₀ 68.5?μM). Quercetin stimulated HSC apoptosis and the apoptotic rate reached 40% at a concentration of 40?μM (EC₅₀ 51.6?μM). Quercetin induced downregulation of Bcl-2 and upregulation of Bax, and increased cytochrome C in the cytoplasm in HSCs. The cleaved forms of caspase-9, caspase-3 and PARP-1 were also increased by quercetin. Furthermore, quercetin elevated mRNA and protein expression of calnexin and CHOP in HSCs but not in hepatocytes. Quercetin also increased phosphorylation of PERK and IRE1 and ATF6 cleavage. However, ERS inhibitor salubrinal significantly abrogated quercetin induction of HSC apoptosis.

Conclusion: Quercetin activated ERS pathway in HSCs leading to apoptosis. We characterized an ERS-mediated mechanism for quercetin as a promising antifibrotic agent.     

Dihydrotanshinone induces apoptosis of SGC7901 and MGC803 cells via activation of JNK and p38 signalling pathways

Context: Dihydrotanshinone (DHT), a natural compound from Salvia miltiorrhiza Bunge (Lamiaceae), showed higher cytotoxic potential compared with other tanshinones. Its effect and mechanism on gastric cancer have not been investigated.

Objective: This study evaluates the effects of DHT on cell proliferation and apoptosis on gastric cancer cells, and elucidates its molecular mechanisms.

Materials and methods: Human gastric cancer MGC803 and SGC7901 cells were treated with various concentrations of DHT (0–15?μM) for 24 and 48?h, and cell growth was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Cell apoptosis was analysed by flow cytometry and DAPI staining. Western blots were performed to investigate changes in the level of apoptosis related genes in gastric cancer cell.

Results: DHT exhibited obvious inhibition of the survival of gastric cancer cells. The IC₅₀ values in SGC7901 and MGC803 cells were 9.14 and 5.39?μM for 24?h, respectively. Cells treated with 6?μM DHT resulted in 41.3% and 35.4% apoptotic cell fractions in SGC7901 and MGC803 cells, respectively, significantly higher than that of the control. Hallmarks of apoptosis were observed in gastric cancer cells after DHT exposure. DHT enhanced the expression levels of cleaved caspase-3, caspase-9 and poly-ADP-ribose polymerases. Furthermore, DHT increased the phosphorylation of JNK and p38 in SGC7901 and MGC803 cells.

Conclusion: DHT induced growth inhibition and apoptosis of gastric cancer cells, involving activation of caspase proteins and the JNK/p38 signaling pathway. The results indicated that DHT has a promising chemotherapeutic potential for human gastric cancer.     

三氧化二砷复合物抑制人脑胶质瘤U87细胞生长的机制研究

目的 研究三氧化二砷复合物对人脑胶质瘤U87细胞凋亡的影响，并进一步探讨其作用机制。方法 采用激光共聚焦技术研究三氧化二砷各复合物进入细胞的方式；采用细胞划痕愈合试验观察其对U87细胞迁移能力的影响；采用血管形成试验观察其对新生血管形成能力的影响；流式细胞仪检测各组细胞周期及细胞凋亡；免疫蛋白印迹法检测凋亡相关蛋白Bcl-2、Bax和caspase-3的表达情况。结果 三氧化二砷复合物主要通过内吞方式进入细胞，能显著抑制U87细胞的迁移以及新生血管的形成；其可诱导U87细胞周期阻滞于G2/M期，促进细胞发生凋亡；三氧化二砷复合物促进Bax和caspase-3的表达，抑制Bcl-2的表达。结论 三氧化二砷复合物对U87细胞的抑制作用机制可能是通过内吞方式进入细胞后，上调Bax的表达，下调Bcl-2的表达，最终诱导细胞发生凋亡。     

Protective effects of Semiaquilegia adoxoides n-butanol extract against hydrogen peroxide-induced oxidative stress in human lens epithelial cells

Context Hydrogen peroxide (H₂O₂)-induced damage in the lens epithelium leads to cell death and cataract. Semiaquilegia adoxoides (DC.) Makino (Ranunculaceae), a folk medicine of Hmong (an ethnic group of China), has been traditionally used to treat cataract; however, the underlying molecular mechanism is yet to be uncovered.

Objective This study aimed to investigate whether the n-butanol extract of S. adoxoides (nSA) is effective against the H₂O₂-induced oxidative stress in human lens epithelial (HLE) cells.

Materials and methods Human lens epithelial (SRA 01/04) cells were stimulated by H₂O₂ (250?μM) in the presence or absence of nSA. The antioxidant effects of nSA were determined in terms of cell viability (MTT assay), apoptosis (AnnexinV/PI staining), radical scavenging capability (various enzymatic assays), loss of mitochondrial membrane potential (Rhodamine 123 staining), expression of apoptotic markers including caspase-3 and caspase-9 and the change of Bcl-2/Bax ratio (western blot) in the HLE cells.

Results The results showed that pretreatment of nSA (250, 500 and 1000?μg/mL) markedly reduced H₂O₂-induced cellular apoptosis and malondialdehyde accumulation, but elevated the activities of total superoxide dismutase, catalase, glutathione peroxidase. Thus, the total antioxidative capability was enhanced upon the nSA treatment meanwhile the loss of mitochondrial membrane potential was prevented. Moreover, nSA at concentrations of 250, 500 and 1000?μg/mL also significantly suppressed the activation of caspase-3 and -9, and increased the Bcl-2/Bax ratio in the HLE cells.

Discussion and conclusion Our findings suggested that nSA is a potential prophylactic agent in the prevention of cataractogeneis.     

氧化槐果碱对胃癌MGC80-3细胞增殖和凋亡的影响

目的 观察氧化槐果碱对胃癌MGC80-3细胞增殖和凋亡的影响及机制。方法 采用CCK-8、Hoechest 33342染色、倒置显微镜和流式细胞术测定氧化槐果碱干预后对细胞的增殖、凋亡的影响,qRT-PCR检测胃癌MGC80-3细胞中Bcl-2、Bax mRNA水平,Western blot法检测胃癌MGC80-3细胞中Bcl-2、Bax、cleaved caspase-3、cleaved caspase-9蛋白水平。结果 氧化槐果碱能抑制胃癌MGC80-3细胞增殖（P<0.05）,并具有时间和浓度依赖性。0.48,0.95,1.91 mmol·L^-1氧化槐果碱能诱导细胞凋亡（P<0.05）,凋亡抑制剂Z-DEVD-FMK能逆转氧化槐果碱的凋亡诱导作用,0.95,1.91 mmol·L^-1氧化槐果碱能上调Bax的mRNA水平,下调Bcl-2的mRNA水平,差异均有统计学意义（P<0.05）。0.95,1.91 mmol·L^-1氧化槐果碱能上调Bax、cleaved caspase-3、cleaved caspase-9的蛋白水平,下调Bcl-2蛋白水平,差异均有统计学意义（P<0.05或P<0.01）。凋亡抑制剂Z-DEVD-FMK能逆转氧化槐果碱对Bcl-2、Bax、cleaved caspase-3、cleaved caspase-9蛋白的影响。结论 氧化槐果碱能通过调节Bcl-2、Bax、cleaved caspase-3、cleaved caspase-9的表达水平,抑制胃癌MGC80-3细胞增殖,诱导细胞凋亡。     

4-Nerolidylcatechol: apoptosis by mitochondrial mechanisms with reduction in cyclin D1 at G0/G1 stage of the chronic myelogenous K562 cell line

Context: 4-Nerolidylcatechol (4-NRC) has showed antitumor potential through apoptosis. However, its apoptotic mechanisms are still unclear, especially in leukemic cells.

Objectives: To evaluate the cytotoxic potential of 4-NRC and its cell death pathways in p53-null K562 leukemic cells.

Materials and methods: Cytotoxicity of 4-NRC (4.17–534.5?μM) over 24?h of exposure was evaluated by MTT assay. 4-NRC-induced apoptosis in K562 cells was investigated by phosphatidylserine (PS) externalization, cell cycle, sub-G1, mitochondrial evaluation, cytochrome c, cyclin D1 and intracellular reactive oxygen species (ROS) levels, and caspase activity analysis.

Results: IC₅₀ values obtained were 11.40, 27.31, 15.93 and 15.70?μM for lymphocytes, K562, HL-60 and Jurkat cells, respectively. In K562 cells, 4-NRC (27?μM) promoted apoptosis as verified by cellular morphological changes, a significant increase in PS externalization and sub-G1 cells. Moreover, it significantly arrested the cells at the G0/G1 phase due to a reduction in cyclin D1 expression. These effects of 4-NRC also significantly promoted a reduction in mitochondrial activity and membrane depolarization, accumulation of cytosolic cytochrome c and ROS overproduction. Additionally, it triggered an increase in caspases -3/7, -8 and -9 activities. When the cells were pretreated with N-acetyl-l-cysteine ROS scavenger, 4-NRC-induced apoptosis was partially blocked, which suggests that it exerts cytotoxicity though not exclusively through ROS-mediated mechanisms.

Discussion and conclusion: 4-NRC has antileukemic properties, inducing apoptosis mediated by mitochondrial-dependent mechanisms with cyclin D1 inhibition. Given that emerging treatment concepts include novel combinations of well-known agents, 4-NRC could offer a promising alternative for chemotherapeutic combinations to maximize tumour suppression.     

Naringenin-induced apoptosis is attenuated by Bcl-2 but restored by the small molecule Bcl-2 inhibitor,HA 14-1, in human leukemia U937 cells

Naringenin, a naturally occurring citrus flavonone, has shown cytotoxicity in various human cancer cell lines as well as inhibitory effects on tumor growth and there is increasing interest in its therapeutic applications. In this study, the effect of ectopic Bcl-2 expression on naringenin-induced apoptosis was investigated. We found that Bcl-2 overexpression markedly protected human leukemia U937 cells from time- and dose-dependent induction of apoptosis by naringenin, as did caspase-3 and caspase-9 inhibitors. Additionally, Bcl-2 overexpression attenuated naringenin-induced Bax translocation and cytosolic release of cytochrome c. Our results also indicated that co-administration of HA14-1 and naringenin increased apoptosis in Bcl-2 overexpressing U937 cells by restoring mitochondrial dysfunction and activation of caspase-9 and caspase-3, as well as by cleavage of poly (ADP-ribose) polymerase. Taken together, these observations indicate that Bcl-2 confers apoptosis resistance to naringenin by inhibiting a mitochondrial amplification step in U937 cells.     

Leonurine hydrochloride induces apoptosis of H292 lung cancer cell by a mitochondria-dependent pathway

Abstract

Context: Leonurine hydrochloride (LH), a major alkaloid compound extracted from Leonurus japonicas Houtt. (Labiatae), is considered to have antitumor roles.

Objective: This study investigated its effects on human non-small cell lung cancer (NSCLC) H292 cells and illustrated the possible mechanism involved.

Materials and methods: After treatment with different concentrations of LH (0, 10, 25, and 50?μmol/L) for 6, 12, 24, 48, and 72?h, the cell viability was assessed by the MTT assay. After exposed to different doses of LH for 24?h, cell-cycle distribution, cell apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were monitored by flow cytometry. RT-PCR and western blot were used to detect the expression of apoptosis-related genes.

Results: LH significantly inhibited the proliferation of H292 cells in a time- and dose-dependent manner, and induced G0/G1 cell-cycle arrest. Coincidentally, LH treatment at a dose of 10, 25, and 50?μmol/L for 24?h increased apoptotic ratio from 4.9?±?0.43% to 11.5?±?1.12%, 19.3?±?1.16%, and 61.3?±?6.69%, respectively. The inhibition effect of LH on H292 cells was associated with the loss of MMP and the generation of ROS. The phosphorylation level of p38 was increased and Akt phosphorylation was reduced by LH treatment. Furthermore, LH treatment increased the expression levels of caspase-3, caspase-9 and Bax/Bcl-2.

Conclusions: LH inhibits the proliferation and induces the apoptosis of H292 cells in a mitochondria-dependent pathway, and the specific mechanism need to be further explored.     

Goniothalamin-induced oxidative stress,DNA damage and apoptosis via caspase-2 independent and Bcl-2 independent pathways in Jurkat T-cells

Goniothalamin (GTN) isolated from Goniothalamus sp. has been demonstrated to induce apoptosis in a variety of cancer cell lines including Jurkat T leukemia cells. However, the mechanism of GTN-induced apoptosis upstream of mitochondria is still poorly defined. In this study, GTN caused a decrease in GSH with an elevation of reactive oxygen species as early as 30 min and DNA damage as assessed by Comet assay. Analysis using topoisomerase II processing of supercoiled pBR 322 DNA showed that GTN caused DNA damage via a topoisomerase II-independent pathway suggesting that cellular oxidative stress may contribute to genotoxicity. A 12-fold increase of caspase-2 activity was observed in GTN-treated Jurkat cells after 4 h treatment and this was confirmed using Western blotting. Although the caspase-2 inhibitor Z-VDVAD-FMK inhibited the proteolytic activity of caspase-2, apoptosis ensued confirming that caspase-2 activity was not crucial for GTN-induced apoptosis. However, GTN-induced apoptosis was completely abrogated by N-acetylcysteine further confirming the role of oxidative stress. Since cytochrome c release was observed as early as 1 h without any appreciable change in Bcl-2 protein expression, we further investigated whether overexpression of Bcl-2 confers resistance in GTN-induced cytotoxicity. Using a panel of Jurkat Bcl-2 transfectants, GTN cytotoxicity was not abrogated in these cells. In conclusion, GTN induces DNA damage and oxidative stress resulting in apoptosis which is independent of both caspase-2 and Bcl-2.     

Anticancer activity of Saussurea lappa extract by apoptotic pathway in KB human oral cancer cells

Abstract

Context: Saussurea lappa Dence (Compositae) is used as a traditional herbal medicine to treat abdominal pain and tenesmus in East Asia. Current studies have shown that S. lappa has anticancer activity in divergent of cancer cells. However, the effects of S. lappa on oral cancer and its mechanisms of action have yet to be elucidated.

Objective: To explore its potential chemotherapeutic effects and mechanism of cell growth inhibition on human oral cancer cells.

Materials and methods: The dried roots of S. lappa were used in this study. Cell viability of KB cells was evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay after treatment with 30?µg/ml of methanol extract from the dried roots of S. lappa. To understand whether its effect on cell death is related with apoptosis pathway, we performed DNA fragmentation assay, western blot, caspase activity assay and fluorescence-activated cell sorting (FACS) analysis.

Results: Treatment of S. lappa extract onto KB cells reduced cell viability significantly with an IC₅₀ value of 30?µg/ml. The formation of a DNA ladder was observed starting at the 24?h treatment. In western blotting analysis, the S. lappa extract induced the proteolytic processing of caspase-3, -9 and poly (ADP-ribose) polymerase, a significant increase of Bax and marked reduction of Bcl-2. We also confirmed the activation of caspase-3/-7 in living KB cells by fluorescence microscopy.

Conclusion: These results suggested that S. lappa extract inhibited cell proliferation through the apoptosis pathway in KB human oral cancer cells.     

Intrinsic pathway of hydroquinone induced apoptosis occurs via both caspase-dependent and caspase-independent mechanisms

The role of mitochondria and apical caspases in apoptosis induced by the benzene metabolite hydroquinone (HQ) remains to be elucidated. Here, we investigated the involvement of mitochondria and activation of the apical caspases-8 and -9 in HQ induced apoptosis in myeloperoxidase (MPO)-rich HL-60 and MPO-deficient Jurkat T cells. Treatment of HL-60 and Jurkat cells with HQ resulted in apoptosis as assessed by phosphatidyl serine (PS) exposure, loss of mitochondrial transmembrane potential (MTP), release of cytochrome c, and processing of apical caspases-8 and -9 and executioner caspase-3. In HQ-treated HL-60 cells, pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (ZVAD), which did not inhibit PS exposure, also failed to abrogate the loss of MTP and release of cytochrome c. However, complete processing of caspase-9 was inhibited in the presence of ZVAD. In marked contrast, in HQ-treated Jurkat cells, ZVAD significantly abrogated PS exposure, loss of MTP, and caspase-9 processing but not release of cytochrome c. Although ZVAD-sensitive caspase-8 processing occurred in both cell types, pretreatment with either fas-receptor blocking ZB4 or fas-ligand NOK1 neutralizing antibodies did not inhibit HQ-induced apoptosis. In conclusion, our results demonstrate that HQ induced apoptosis in Jurkat cells occurs via a ZVAD-inhibitable, caspase-dependent process, while in HL-60 cells, apoptosis occurs predominantly via caspase-independent mechanisms. Our results emphasize that both caspase-dependent and independent mechanisms should be considered in the intrinsic apoptotic pathway induced by HQ.     

Dexamethasone-induced apoptosis in PC12 cells

Abstract

This study aims to evaluate the cytotoxicity and damage of dexamethasone (DEX) on rat pheochromocytoma (PC12) cells by determining cell viability, Hoechst 33342 staining, mitochondrial depolarization assays, opened mitochondrial permeability transition pores (MPTPs) detection, and measurement of Caspase-3 and Bcl-2 activities. The results show that DEX inhibits PC12 cell growth and decreases their viability in a remarkable dose-dependent manner. Our results also reveal that DEX exposure causes morphologic changes, opening of MPTPs and mitochondrial depolarization, upregulates Caspase-3 expression, and suppresses Bcl-2 expression in PC12 cells. These results suggest that DEX-induced apoptosis may be mediated through mitochondrial dysfunction.     

Peptides extracted from edible mushroom: Lentinus squarrosulus induces apoptosis in human lung cancer cells

Context: Lentinus squarrosulus Mont. (Polyporaceae) is an interesting source of diverse bioactive compounds.

Objective: This is the first study of the anticancer activity and underlying mechanism of peptides extracted from Lentinus squarrosuls.

Materials and methods: Peptides were isolated from the aqueous extract of L. squarrosulus by employing solid ammonium sulphate precipitation. They were further purified by ion-exchange chromatography on diethylaminoethanol (DEAE)-cellulose and gel filtration chromatography on Sephadex G25. Anticancer activity was investigated in human lung cancer H460, H292 and H23 cells cultured with 0–40?μg/mL of peptide extracts for 24?h. Cell viability and mode of cell death were evaluated by MTT and nuclear staining assay, respectively. Western blotting was used to investigate the alteration of apoptosis-regulating proteins in lung cancer cells treated with peptide extracts (0–20?μg/mL) for 24?h.

Results: The cytotoxicity of partially-purified peptide extracts from L. squarrosulus was indicated with IC₅₀ of ～26.84?±?2.84, 2.80?±?2.14 and 18.84?±?0.30?μg/mL in lung cancer H460, H292 and H23 cells, respectively. The extracts at 20?μg/mL induced apoptosis through the reduction of anti-apoptotic Bcl-2 protein (～0.5-fold reduction) and up-regulation of BAX (～4.5-fold induction), a pro-apoptotic protein. Furthermore, L. squarrosulus peptide extracts (20?μg/mL) also decreased the cellular level of death receptor inhibitor c-FLIP (～0.6-fold reduction).

Conclusions and discussion: This study provides the novel anticancer activity and mechanism of L. squarrosulus peptide extracts, which encourage further investigation and development of the extracts for anticancer use.