Unveiling the neuroprotection effects of Volvalerenic acid A: Mitochondrial fusion induction via IDO1-mediated Stat3-Opa1 signaling pathway.

BACKGROUND: Ischemic stroke is a leading cause of death and long-term disability worldwide. Studies have suggested that cerebral ischemia induces massive mitochondrial damage. Valerianic acid A (VaA) is the main active ingredient of valerianic acid with neuroprotective activity. PURPOSE: This study aimed to investigate the neuroprotective effects of VaA with ischemic stroke and explore the underlying mechanisms. METHOD: In this study, we established the oxygen-glucose deprivation and reperfusion (OGD/R) cell model and the middle cerebral artery occlusion and reperfusion (MCAO/R) animal model in vitro and in vivo. Neurological behavior score, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and Hematoxylin and Eosin (HE) Staining were used to detect the neuroprotection of VaA in MCAO/R rats. Also, the levels of ROS, mitochondrial membrane potential (MMP), and activities of NAD+ were detected to reflect mitochondrial function. Mechanistically, gene knockout experiments, transfection experiments, immunofluorescence, DARTS, and molecular dynamics simulation experiments showed that VaA bound to IDO1 regulated the kynurenine pathway of tryptophan metabolism and prevented Stat3 dephosphorylation, promoting Stat3 activation and subsequent transcription of the mitochondrial fusion-related gene Opa1. RESULTS: We showed that VaA decreased the infarct volume in a dose-dependent manner and exerted neuroprotective effects against reperfusion injury. Furthermore, VaA promoted Opa1-related mitochondrial fusion and reversed neuronal mitochondrial damage and loss after reperfusion injury. In SH-SY5Y cells, VaA (5, 10, 20 µM) exerted similar protective effects against OGD/R-induced injury. We then examined the expression of significant enzymes regulating the kynurenine (Kyn) pathway of the ipsilateral brain tissue of the ischemic stroke rat model, and these enzymes may play essential roles in ischemic stroke. Furthermore, we found that VaA can bind to the initial rate-limiting enzyme IDO1 in the Kyn pathway and prevent Stat3 phosphorylation, promoting Stat3 activation and subsequent transcription of the mitochondrial fusion-related gene Opa1. Using in vivo IDO1 knockdown and in vitro IDO1 overexpressing models, we demonstrated that the promoted mitochondrial fusion and neuroprotective effects of VaA were IDO1-dependent. CONCLUSION: VaA administration improved neurological function by promoting mitochondrial fusion through the IDO1-mediated Stat3-Opa1 pathway, indicating its potential as a therapeutic drug for ischemic stroke.

Ginseng saponin metabolite 20(S)-protopanaxadiol relieves pulmonary fibrosis by multiple-targets signaling pathways.

Background: Panax ginseng Meyer is a representative Chinese herbal medicine with antioxidant and anti-inflammatory activity. 20(S)-Protopanaxadiol (PPD) has been isolated from ginseng and shown to have promising pharmacological activities. However, effects of PDD on pulmonary fibrosis (PF) have not been reported. We hypothesize that PDD may reverse inflammation-induced PF and be a novel therapeutic strategy. Methods: Adult male C57BL/6 mice were used to establish a model of PF induced by bleomycin (BLM). The pulmonary index was measured, and histological and immunohistochemical examinations were made. Cell cultures of mouse alveolar epithelial cells were analyzed with Western blotting, co-immunoprecipitation, immunofluorescence, immunohistochemistry, siRNA transfection, cellular thermal shift assay and qRT-PCR. Results: The survival rate of PPD-treated mice was higher than that of untreated BLM-challenged mice. Expression of fibrotic hallmarks, including α-SMA, TGF-ß1 and collagen I, was reduced by PPD treatment, indicating attenuation of PF. Mice exposed to BLM had higher STING levels in lung tissue, and this was reduced by phosphorylated AMPK after activation by PPD. The role of phosphorylated AMPK in suppressing STING was confirmed in TGF-ß1-incubated cells. Both in vivo and in vitro analyses indicated that PPD treatment attenuated BLM-induced PF by modulating the AMPK/STING signaling pathway. Conclusion: PPD ameliorated BLM-induced PF by multi-target regulation. The current study may help develop new therapeutic strategies for preventing PF.

Isofraxidin enhances hyperthermia­induced apoptosis via redox modification in acute monocytic leukemia U937 cells.

The cell­killing potential of most chemotherapeutic agents is enhanced by a temperature elevation. Isofraxidin (IF) is a coumarin compound widely found in plants, such as the Umbelliferae or Chloranthaceae families. IF induces anticancer effects in lung and colorectal cancer. To the best of our knowledge, the combined effects of hyperthermia (HT) and IF on heat­induced apoptosis have not been reported. Acute monocytic leukemia U937 cells were exposed to HT with or without IF pre­treatment. Apoptosis was measured by Annexin V­FITC/PI double staining assay using flow cytometry and cell viability was observed by cell counting kit assay, DNA fragmentation. The mechanism involved in the combination was explored by measuring changes in the mitochondrial membrane potential, (MMP), intracellular ROS generation, expression of apoptosis related protein, and intracellular calcium ion level. It was demonstrated that IF enhanced HT­induced apoptosis in U937 cells. The results demonstrated that combined treatment enhanced mitochondrial membrane potential loss and transient superoxide generation increased protein expression levels of caspase­3, caspase­8 and phosphorylated­JNK and intracellular calcium levels. Moreover, the role of caspases and JNK was confirmed using a pan caspase inhibitor (zVAD­FMK) and JNK inhibitor (SP600125) in U937 cells. Collectively, the data demonstrated that IF enhanced HT­induced apoptosis via a reactive oxygen species mediated mitochondria/caspase­dependent pathway in U937 cells.

Spatiotemporal evolution of nutrients and the influencing factors in Laizhou Bay over the past 40 years.

The nutrient status in Laizhou Bay has changed in composition and structure as a result of anthropogenic activities and climate change, which has led to several environmental problems (e.g., eutrophication, organic pollution and red tides). To better understand the spatiotemporal variations in dissolved inorganic nitrogen (DIN), dissolved inorganic phosphorus (DIP), and eutrophication in the Laizhou Bay, we collected historical research data and conducted four cruises in 2021. The highest surface DIN was found to occur in autumn and predominantly concentrated in the southwestern bay. The highest surface DIP content was found in winter and distributed in the northwestern bay. Surface organic pollution showed estuaries as the most polluted areas. In the past 40-60 years, the DIN, DIP, and eutrophication have shown an inverted U-shaped trend, and the bay has changed from N limitation to P limitation. Economic development, phytoplankton absorption, and bottom mineral release are factors influencing the content and distribution of nutrient in the bay.

Ecological risk assessment of heavy metal pollutants and total petroleum hydrocarbons in sediments of the Bohai Sea, China.

Heavy metals and organic pollutants like total petroleum hydrocarbons (TPHs) in coastal marine sediments are receiving extensive attention, as they may pose a serious threat to the aquatic environment and ecosystem health. To date, however, data on the long-term variations in the levels of sedimentary heavy metals and TPHs as well as their ecological risks are relatively limited. Here, we conducted 12 cruises spanning 3 years in the Bohai Sea and obtained ~1400 sediment samples to explore the long-term variations of heavy metals (i.e., Hg, As, Cu, Zn, Pb, Cd) and TPHs, and to assess their potential ecological risks. The results suggested that the ranges for the levels of Hg, As, Cu, Zn, Pb, Cd, and TPHs in sediments between 2019 and 2021 were <0.01-0.07, 0.23-10.72, 8.07-20.67, 25.52-46.55, 10.94-28.19, 0.14-0.56, and 9.14-18.41 mg kg-1, respectively. Based on the single factor evaluation (Fi) for sediment quality, we found that most of the evaluation factors in the study area met the requirements of sediment quality standard (i.e., Fi < 1), except for the factor of metal Cd in some cases. The implication is that the sediment in the Bohai Sea was fairly clean in terms of heavy metals and TPHs. However, the concentration of metal Cd exceeded the sediment quality standard during May 2019 and 2020 (i.e., Fi > 1), indicating that Cd could be identified as a major pollutant in surface sediments. Also, based on the ecological risk assessment (Ei) of heavy metal pollutants, we found that the metal Cd had reached a level with potential ecological risk in some cases (80 ≤ Ei < 160). As such, we further suggested that the Cd contamination might have a potential risk on the Bohai Sea' ecosystem.

Cordycepin enhances hyperthermia-induced apoptosis and cell cycle arrest by modulating the MAPK pathway in human lymphoma U937 cells.

BACKGROUND: Hyperthermia induces cancer cell death. However, the cytotoxic effect of hyperthermia is not sufficient. Cordycepin can also induce apoptosis in cancer cells and enhance the antitumoral activity of irradiation. To examine cordycepin-mediated enhancement of hyperthermia-induced apoptosis, this study investigated the combined effects and apoptotic mechanisms of hyperthermia and cordycepin on human leukemia U937 cells. METHODS: Cell viability and apoptosis were measured using MTT assays, Hoechst 33342 staining and Annexin V/PI double staining. The distribution of the cell cycle and sub-G1 phase, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were examined by flow cytometry. The expression of related proteins was analyzed by western blotting. RESULTS: Combined treatment with hyperthermia and cordycepin markedly augmented apoptosis by upregulating Bax and suppressing Bcl-2, Bid and activated caspase 3 and 8 expression, and apoptosis was decreased by Z-VAD-fmk (a pan caspase inhibitor). We also found that the MMP was significantly decreased and excessive ROS generation occurred. The combination treatment also induced arrest in the G2/M phase by downregulating cyclin dependent kinase 1 (CDK1) and cyclin B1 protein expression. Furthermore, it was observed that mitogen-activated protein kinase (MAPK) pathway including ERK, JNK and p38 signals was involved in the induction of apoptosis. The phosphorylated p38 and JNK were increased and ERK phosphorylation was decreased by the combined treatment. In addition, N-acetyl-L-cysteine (NAC) significantly protected the cells by restoring ROS levels and the activity of caspase-3, inactivating the MAPK pathway. CONCLUSION: Cordycepin significantly enhanced hyperthermia-induced apoptosis and G2/M phase arrest in U937 cells. The combined treatment enhanced apoptosis through the MAPK pathway and mitochondrial dysfunction, and these effects could be rescued by NAC. We report for the first time that cordycepin can be used as a hyperthermia sensitizer to treat leukemia.

Jiuzhuan Huangjing Pills relieve mitochondrial dysfunction and attenuate high-fat diet-induced metabolic dysfunction-associated fatty liver disease.

BACKGROUND: Metabolic dysfunction-associated fatty liver disease (MAFLD) is a common global chronic liver disease. Jiuzhuan Huangjing Pills (JHP) have been used for the treatment of human disease for over a thousand years, but their efficacy and underlying mechanism(s) of action against MAFLD are unknown. We investigated the alleviating effects of JHP on high-fat diet (HFD)-induced MAFLD. METHODS: In vitro and in vivo methods were used to evaluate the effects of JHP on MAFLD. L02 adipocyte models were induced by fat emulsion and adipocytes were treated with JHP for 24 h. MAFLD rat models were induced by HFD-feeding and were intragastrically administered JHP for 12 weeks. Changes in fat accumulation, L02 cell damage, body weight, food intake, histological parameters, organ indexes, biochemical parameters, and mitochondrial indicators including ultrastructure, oxidative stress, energy metabolism, and fatty acid metabolism were investigated. RESULTS: JHP attenuated the increase in levels of total cholesterol, triglyceride, low density lipoprotein cholesterol, alanine transaminase, and aspartate transaminase levels, and significantly increased high density lipoprotein cholesterol. JHP up-regulated levels of glutathione (GSH) and superoxide dismutase (SOD), and down-regulated malondialdehyde (MDA). JHP afforded protection to the mitochondrial ultrastructure, and inhibited the HFD-induced increase in MDA and the reduction of SOD, GSH, ATP synthase, and complex I and II, in liver mitochondria. JHP regulated the expression of ß-oxidation genes, including acyl-CoA dehydrogenase, cyl-CoA dehydrogenase long chain, carnitine palmitoyltransferase 1A, carnitine palmitoyltransferase 1B, peroxisomal proliferator-activated receptor-gamma coactivator-1α and peroxide proliferator activated receptor α. CONCLUSION: JHP alleviates HFD-induced MAFLD through the protection of mitochondrial function.

Comparative study on protective effect of different selenium sources against cadmium-induced nephrotoxicity via regulating the transcriptions of selenoproteome.

Cadmium (Cd) is a ubiquitous environmental pollutant, which mainly input to the aquatic environment through discharge of industrial and agricultural waste, can be a threat to human and animal health. Selenium (Se) possesses a beneficial role in protecting animals and ameliorating the toxic effects of Cd. However, the comparative antagonistic effects of different Se sources such as inorganic, organic Se and nano-form Se on Cd toxicity are still under-investigated. Hence, the purpose of this study was to evaluate the comparative of Se sources antagonism on Cd-induced nephrotoxicity via oxidative stress and selenoproteome transcription. In the present study, Cd-diet disturbed in the system balance of 5 trace elements (Zinc (Zn), copper (Cu), Iron (Fe), Se, Cd) and impaired renal function. Se sources, including nano- Se (NS), Se- yeast (SY), sodium selenite (SS) and mixed selenium (MS) significantly recovered the balance of 4 trace elements (Zn, Cu, Cd, Se) and renal impaired indexes (blood urea nitrogen (BUN) and creatinine (CREA)). Histological appearance of Cd-treated kidney indicated renal tubular epithelial vacuoles, particle degeneration and enlarged capsular space. Ultrastructure observation results illustrated that Cd-induced mitochondrial cristae reduction, membrane disappearance, and nuclear deformation. Treatment with Se sources, NS appeared a better impact on improving kidney tissues against the pathological alterations resulting from Cd administration. Meanwhile, NS reflected a significant impact on relieving Cd-induced kidney oxidative damage, and significantly restored the antioxidant defense system of the body. Our findings also showed NS ameliorated the Cd-induced downtrends expression of selenoproteome and selenoprotein synthesis related transcription factors. Overall, NS was the most effective Se source in avoiding of Cd cumulative toxicity, improving antioxidant capacity and regulating of selenoproteome transcriptome and selenoprotein synthesis related transcription factors expression, which contributes to ameliorate Cd-induced nephrotoxicity in chickens. These results demonstrated diet supplement with NS may prove to be an effective approach for alleviating Cd toxicity and minimizing Cd -induced health risk.

Melatonin sensitises shikonin-induced cancer cell death mediated by oxidative stress via inhibition of the SIRT3/SOD2-AKT pathway.

Recent research suggests that melatonin (Mel), an endogenous hormone and natural supplement, possesses anti-proliferative effects and can sensitise cells to anti-cancer therapies. Although shikonin (SHK) also possesses potential anti-cancer properties, the poor solubility and severe systemic toxicity of this compound hinders its clinical usage. In this study, we combined Mel and SHK, a potentially promising chemotherapeutic drug combination, with the aim of reducing the toxicity of SHK and enhancing the overall anti-cancer effects. We demonstrate for the first time that Mel potentiates the cytotoxic effects of SHK on cancer cells by inducing oxidative stress via inhibition of the SIRT3/SOD2-AKT pathway. Particularly, Mel-SHK treatment induced oxidative stress, increased mitochondrial calcium accumulation and reduced the mitochondrial membrane potential in various cancer cells, leading to apoptosis. This drug combination also promoted endoplasmic reticulum (ER) stress, leading to AKT dephosphorylation. In HeLa cells, Mel-SHK treatment reduced SIRT3/SOD2 expression and SOD2 activity, while SIRT3 overexpression dramatically reduced Mel-SHK-induced oxidative stress, ER stress, mitochondrial dysfunction and apoptosis. Hence, we propose the combination of Mel and SHK as a novel candidate chemotherapeutic regimen that targets the SIRT3/SOD2-AKT pathway in cancer.

Melatonin triggers the anticancer potential of phenylarsine oxide via induction of apoptosis through ROS generation and JNK activation.

Melatonin, a safe endogenous hormone and a natural supplement, has recently been recognized to have antiproliferative effects and the ability to sensitize cells to other anticancer therapies. Phenylarsine oxide (PAO) has anticancer potential but it is considered as a toxic agent. In this study we combined melatonin to reduce the toxicity while securing the anti-cancer effects of PAO. Cell viability was determined by MTT assay, whereas cytotoxic assays were performed using an LDH cytotoxicity assay kit. Cell cycle analysis, Annexin V/PI staining, the mitochondrial membrane potential (MMP), mitochondrial calcium and reactive oxygen species (ROS) generation were analyzed using flow cytometry. Sytox stained cells were visualized by fluorescence microscopy and the expression of proteins was detected by western blotting. Melatonin increased the anticancer potential of PAO by decreasing the cell viability and increasing LDH release in various cancer cells. The mode of cell death was determined to be typical apoptosis, as evidenced by Annexin V/PI-stained cells, PARP cleavage, and caspase-3 activation, and with significant modulations in the expression of proapoptotic, antiapoptotic and cell cycle-related proteins. ROS generation played a critical role in induction of cell death by this combined treatment, which is validated by reversal of cytotoxicity upon cotreatment with NAC. Furthermore, the activation of MAPKs, especially JNK, contributed to the induction of cell death, accompanied by endoplasmic reticulum stress and autophagy, affirmed by the abrogation of cytotoxicity after JNK-IN-8 and TUDCA application. Melatonin showed promising potential as a chemotherapeutic agent in combination with PAO to achieve a better anticancer response.

Protective effect of dihydromyricetin on hyperthermia-induced apoptosis in human myelomonocytic lymphoma cells.

Dihydromyricetin (DMY) is a traditional herbal medicine, with a wide range of biological activities. Extreme hyperthermia (HT) can suppress the immune system; thus, protection of the immune system is beneficial in heat-related diseases, including heatstroke. In our study, we revealed the protective effect of DMY against HT-induced apoptosis and analysed the underlying molecular mechanisms. We incubated human myelomonocytic lymphoma U937 cells at 44 °C for 30 min with or without DMY and followed by further incubation for 6 h at 37 °C. Cell viability was determined by the CCK-8 assay. DMY did not cause any cytotoxic effects in U937 cells even at high doses. HT treatment alone induced significant apoptosis, which was detected by DNA fragmentation and Annexin V/PI double staining. Mitochondrial dysfunction was identified by loss of mitochondrial membrane potential (MMP) during heat stimulation. Apoptotic related proteins were involved, truncated Bid and caspase-3 were upregulated, and Mcl-1 and XIAP were downregulated. We also identified the related signalling pathways, such as the MAPK and PI3K/AKT pathways. However, changes in HT were dramatically reversed when the cells were pretreated with DMY before exposure to HT. Overall, MAPKs and PI3K/AKT signalling, mitochondrial dysfunction, and caspase-mediated pathways were involved in the protective effect of DMY against HT-induced apoptosis in U937 cells, which was totally reversed by DMY pretreatment. These findings indicate a new clinical therapeutic strategy for the protection of immune cells during heatstroke.

Mechanistic study of nonivamide enhancement of hyperthermia-induced apoptosis in U937 cells.

Hyperthermia is one therapeutic tool for damaging and killing cancer cells, with minimal injury to normal tissues. However, its cytotoxic effects alone are insufficient for quantitative cancer cell death. To overcome this limitation, several studies have explored non-toxic enhancers for hyperthermia-induced cell death. Capsaicin may be applicable as a therapeutic tool against various types of cancer. In the present study, we employed nonivamide, a less-pungent capsaicin analogue, to investigate its possible enhancing effects on hyperthermia-induced apoptosis; moreover, we analyzed its molecular mechanism. Treatment of U937 cells at 44 °C for 15 min, combined with nonivamide 50 µM, revealed enhancement of apoptosis. Significant increases in reactive oxygen species generation, mitochondrial dysfunction, and cleaved caspase-3 were observed during the combined treatment; these were accompanied by an increase in pro-apoptotic Bcl-2 family proteins and a decrease in anti-apoptotic Bcl-2 proteins. In addition, significant increases in p-JNK and p-p38 were detected, following the combined treatment. In conclusion, nonivamide enhanced hyperthermia-induced apoptosis via a mitochondrial-caspase dependent pathway. The underlying mechanism may include elevation of intracellular reactive oxygen species, mitochondrial dysfunction, and increased activation of JNK and p38.

Baicalin Augments Hyperthermia-Induced Apoptosis in U937 Cells and Modulates the MAPK Pathway via ROS Generation.

BACKGROUND/AIMS: Hyperthermia is a widely used therapeutic tool for cancer therapy and a well-known inducer of apoptosis. Although the flavonoid compound baicalin (BCN) is a potent anticancer agent for several human carcinomas, it is less potent in the human U937 myelomonocytic leukemia cell line. To explore any enhancing effects of BCN on hyperthermia-induced apoptosis, this study investigated the combined effects and apoptotic mechanisms of hyperthermia and BCN in U937 cells. METHODS: U937 cells were heat treated at 44ºC for 12 min with or without pre-treatment with BCN (10-50 µM) and then incubated for 6 h at 37 ºC with 5% CO2 and 95% air. Cell viability was analyzed by Trypan blue exclusion assay. Apoptosis was examined by DNA fragmentation, fluorescence microscopy and flow cytometry. Generation of mitochondrial trans-membrane potential (MMP), mitochondrial calcium, and reactive oxygen species (ROS) was also detected by flow cytometry. The expression of proteins related to apoptosis and signaling pathways was determined by western blotting. RESULTS: Hyperthermia alone did not reduce cell viability or induce notable levels of apoptosis, but combined hyperthermia and BCN treatment markedly augmented apoptosis by upregulating proapoptotic proteins and suppressing antiapoptotic proteins, culminating in caspase-3 activation. Mitochondrial transmembrane potential was significantly decreased, and generation of reactive oxygen species (ROS) and suppression of antioxidant enzymes were marked. Furthermore, with the combined treatment, the phosphorylated forms of JNK and p38 showed increased expression, whereas AKT was dephosphorylated. JNK-IN-8 (a JNK inhibitor) and NAC (a ROS scavenger) abrogated the apoptotic effects of the combined treatment, significantly protecting the cells and indicating the involvement of high ROS generation and the MAPK pathway in the underlying molecular mechanism. CONCLUSION: This study provides compelling evidence that hyperthermia, in combination with BCN, is a promising therapeutic strategy for enhancement of apoptosis and suggest a promising therapeutic approach for cancer.

Molecular mechanisms of hyperthermia-induced apoptosis enhanced by docosahexaenoic acid: implication for cancer therapy.

To develop a non-toxic enhancer for hyperthermia-induced cell death as a potential cancer treatment, we studied the effect and mechanism of docosahexaenoic acid (DHA) on hyperthermia-induced apoptosis. Treatment with 20µM DHA and 44°C for 10min induced significant apoptosis, increased intracellular reactive oxygen species (ROS), and caspase-3 activation in U937 cells, but heat or DHA alone did not induce notable apoptosis. Decreased mitochondrial transmembrane potentials were dramatically increased by the combined treatment, accompanied by increased pro-apoptotic Bcl-2 family protein tBid, and decreased anti-apoptotic Bcl-2 and Bcl-xL. Combined hyperthermia-DHA treatment induced significant phosphorylation of protein kinase C (PKC)-δ (p-PKC-δ), and apoptosis in a DHA dose-dependent manner. Using both 20µM DHA and 44°C for 10min induced significant PKC-δ cleavage and its translocation to mitochondria. These results were also seen in HeLa cells. However, MAPKs and Akt were not affected by the treatment. In conclusion, DHA enhances hyperthermia-induced apoptosis significantly via a mitochondria-caspase-dependent pathway; its underlying mechanism involves elevated intracellular ROS, mitochondria dysfunction, and PKC-δ activation.

Molecular mechanisms of hyperthermia-induced apoptosis enhanced by withaferin A.

Hyperthermia is a good therapeutic tool for non-invasive cancer therapy; however, its cytotoxic effects are not sufficient. In the present study, withaferin A (WA), a steroidal lactone derived from the plant Withania somnifera Dunal, has been investigated for its possible enhancing effects on hyperthermia-induced apoptosis. In HeLa cells, treatment with 0.5 or 1.0µM WA at 44°C for 30min induced significant apoptosis accompanied by decreased intracellular GSH/GSSG ratio and caspase-3 activation, while heat or WA alone did not induce such changes. The upregulation in apoptosis was significantly inhibited by glutathione monoethyl ester, a cell permeable glutathione precursor. Mitochondrial transmembrane potentials were dramatically decreased by the combined treatment, with increases in pro-apoptotic Bcl-2-family proteins tBid and Noxa, and downregulation of antiapoptotic Bcl-2 and Mcl-1. Combined treatment with hyperthermia and WA induced significant increases in JNK phosphorylation (p-JNK), and decreases in the phosphorylation of ERK (p-ERK) compared with either treatment alone. These results suggest that WA enhances hyperthermia-induced apoptosis via a mitochondria-caspase-dependent pathway; its underlying mechanism involves elevated intracellular oxidative stress, mitochondria dysfunction, and JNK activation.

The molecular mechanisms and gene expression profiling for shikonin-induced apoptotic and necroptotic cell death in U937 cells.

Shikonin (SHK), a natural naphthoquinone derived from the Chinese medical herb Lithospermum erythrorhizon, induces both apoptosis and necroptosis in several cancer cell lines. However, the detailed molecular mechanisms involved in the initiation of cell death are still unclear. In the present study, caspase-dependent apoptosis was induced by SHK treatment at 1µM after 6h in U937 cells, with increase in DNA fragmentation, generation of intracellular reactive oxygen species (ROS), fraction of cells with low mitochondrial membrane potential (MMP), and in the expression of BH3 only proteins Noxa and tBid. Interestingly, caspase-independent cell death was also detected with SHK treatment at 10µM, observed as increase in SYTOX® Green staining and release of lactate dehydrogenase (LDH). Necrostatin-1 (Nec-1) completely inhibited the SHK-induced leakage of LDH and SYTOX® Green staining. Cell permeable exogenous glutathione (GSH) completely inhibited 1µM SHK-induced apoptosis and converted 10µM SHK-induced necroptosis to apoptosis. Gene expression profiling revealed that 353 genes were found to be significantly regulated by 1µM and 85 genes by 10µM of SHK treatment, respectively. Among these genes, the transcription factor 3 (ATF3) and DNA-damage-inducible transcript 3 (DDIT3) were highly expressed at 1µM of SHK treatment, while tumor necrosis factor (TNF) expression mainly increased at 10µM treatment. These findings provide novel information for the molecular mechanism of SHK-induced apoptosis and necroptosis.