Calcium signaling facilitates chilling- and GA- induced dormancy release in tree peony.

Calcium plays a crucial role in plant growth and development, yet little is known about its function in endodormancy regulation. Tree peony (Paeonia suffruticosa), characterized by compound buds and large flowers, is well-known for its ornamental and medicinal value. To break bud dormancy release is a prerequisite of flowering and forcing culture, particularly during the Spring Festival. In this study, the Ca2+ chelator EGTA and Ca2+ channel blocker LaCl3 were applied, resulting in a significant delay in budburst during both chilling- and gibberellin (GA)- induced dormancy release in a dosage-dependent manner. As expected, the retardation of bud break was recovered by the supplementation of 30 mM CaCl2, indicating a facilitating role of calcium in dormancy release. Accordingly, several calcium-sensor-encoding genes including Calmodulin (CaM) and Ca2+-dependent protein kinases (CDPKs) were significantly up-regulated by prolonged chilling and exogenous GAs. Ultrastructure observations revealed a decline in starch grains and the reopening of transport corridors following prolonged chilling. Calcium deposits were abundant in the cell walls and intercellular spaces at the early dormant stage but were enriched in the cytosol and nucleus before dormancy release. Additionally, several genes associated with dormancy release, including EBB1, EBB3, SVP, GA20ox, RGL1, BG6, and BG9, were differentially expressed after calcium blocking and recovery treatments, indicating that calcium might partially modulate dormancy release through GA and ABA pathways. Our findings provide novel insights into the mechanism of dormancy release and offer potential benefits for improving and perfecting forcing culture technology in tree peonies.

Phytochemical Constituents from the Seeds of Capsella bursa-pastoris and Their Antioxidant Activities.

Phytochemical investigation of 70% EtOH extract of the seeds of Capsella bursa-pastoris led to the isolation of a new cyclobutane organic acid (1), and fourteen known compounds, including two organosulfur compounds (2, 3), two quinonoids (4, 5), five flavonoids (6-10), three sterols (11-13) and two other types (14, 15). The structures of the compounds were elucidated by extensive spectroscopic analyses as well as comparison of their spectroscopic data with those reported in the literature. The antioxidant capacities of all compounds and extractive fractions were evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging test and ferric reducing antioxidant power (FRAP) assay. Then the antioxidative substances were evaluated for their neuroprotective effects against H2O2-induced HT22 cell injury. The results indicated the strong scavenging ability to free radical of the extractive fractions and compounds 1-3, 8-10 and 13, and the ferric reducing antioxidant power of the extractive fractions and compounds 1-3, 8 and 10, which were close to or higher than that of the positive control trolox. The EtOAc fraction, n-BuOH fraction, and compounds 1, 3 and 8 can protect HT-22 cells from oxidative damage.

Overcoming Basal Autophagy, Kangai Injection Enhances Cisplatin Cytotoxicity by Regulating FOXO3a-Dependent Autophagic Cell Death and Apoptosis in Human Lung Adenocarcinoma A549/DDP Cells.

Cisplatin resistance is one of the major obstacles in the treatment of nonsmall cell lung cancer (NSCLC). Kangai injection (KAI), a Chinese herbal medicine, has been used in tumors as adjuvant treatment, but its exact antitumor mechanism is still unclear. In this study, we first demonstrated that cisplatin-resistant A549/DDP cells showed a higher level of basal autophagy in response to cisplatin treatment with increasing autophagic protein expression levels of Beclin 1, p62, and LC3 compared to cisplatin-sensitive A549/DDP cells; then, we assessed the antitumor effect of KAI in cisplatin-resistant lung adenocarcinoma A549/DDP cells. Our results showed that KAI exhibited direct cytotoxic and chemosensitizing effects in A549/DDP cells. Combining KAI with cisplatin promoted A549/DDP cell apoptosis, which was confirmed by cell cycle arrest, condensed nuclear chromatin, annexin V fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining, and apoptosis-related protein expression. In addition, combining KAI with cisplatin induced autophagic cell death in A549/DDP cells with a high level of basal autophagy, as indicated by an increase in LC3 spot count, an accumulation of Beclin 1 and LC3 II, and reduced p62 protein expression. We also found that the apoptosis and autophagic cell death induced by cotreatment of KAI and cisplatin in A549/DDP cells were FOXO3a-dependent as indicated by decreased p-FOXO3a expression and increased FOXO3a nuclear localization, respectively. Furthermore, the FOXO3a gene knockdown assay further confirmed that KAI enhanced cisplatin cytotoxicity in A549/DDP cells with a high level of basal autophagy by inducing apoptosis and autophagic cell death in a FOXO3a-dependent manner. These findings suggest that the combination of KAI and cisplatin might support the potential clinical treatment as a novel strategy to overcome cisplatin resistance.

The effect of transcutaneous electrical nerve stimulation (TENS) on pain control and phenylethanolamine-N-methyltransferase (PNMT) gene expression after cesarean section.

Transcutaneous electrical nerve stimulation (TENS) is one of the non-pharmacological methods of pain relief that has been able to reduce pain by 70 to 90% in postoperative pain control. This study aimed to determine the effect of TENS on pain control after cesarean section and its effect on PNMT gene expression. For this purpose, a double-blind randomized clinical trial was performed on 70 Chinese patients with elective cesarean section. Patients were divided into case and control groups. In the case group, TENS and analgesic drugs were used to relieve pain, and in the control group, the only analgesic drug was used. Then the severity of pain, recurrence of pain attacks, the number of analgesic drugs used and the amount of analgesic drug used in the first 24 hours after surgery were evaluated and compared. Blood samples were also taken from patients to evaluate PNMT gene expression. The semi-quantitative RT-PCR was used to study changes in gene expression. The results showed that the group treated with TENS had less pain intensity and less recurrence of pain attacks than the group that received only analgesic medication. Also, the frequency of analgesic drug use and its dose in the TENS group were significantly lower than in the control group. TENS, on the other hand, has been able to greatly reduce the expression of the PNMT gene, which is produced during times of stress. Therefore, it is recommended that TENS be used as a non-invasive and non-pharmacological adjuvant effective in reducing pain after cesarean section.

Shenmai injection enhances cisplatin-induced apoptosis through regulation of Mfn2-dependent mitochondrial dynamics in lung adenocarcinoma A549/DDP cells.

Aim: Chemoresistance is the biggest obstacle in cancer treatment. Our previous study demonstrated that Shenmai injection (SMI), a Chinese herbal medicine, enhanced the antitumor effect of cisplatin via glucose metabolism reprogramming. This study aimed to further determine whether the SMI sensitizes the non-small cell lung cancer (NSCLC) cells to cisplatin through regulation mitochondrial dynamics. Methods: The Kaplan-Meier Plotter database was used to investigate the relationship between mRNA expression of mitofusin-2 (Mfn2) and the survival analysis of NSCLC patients. The protein expression of Mfn2 in a lung adenocarcinoma tissue chip was detected by immunohistochemistry staining. The expression of Mfn2 and ATAD3A were compared between cisplatin-sensitive A549 and cisplatin-resistant A549/DDP cells. Additionally, A549/DDP cells were co-treated with cisplatin and SMI to detect mitochondrial morphology by fluorescent staining, apoptosis-related protein expression with Western blotting, and mitochondrial membrane potential (ΔΨm) with flow cytometry analysis. Results: The mean survival time of the Mfn2low group was significantly lower than that of the Mfn2high group by Kaplan-Meier Plotter database analysis, and the Mfn2 protein expression level was lower in cancer tissues than in adjacent tissues. The combination of SMI and cisplatin induced dynamic changes in A549/DDP cells, with increased mitochondrial fusion followed by upregulation of Mfn2 and downregulation of ATAD3A and reduced mitochondrial mass and ΔΨm. Moreover, SMI significantly enhanced cisplatin-induced A549/DDP apoptosis, upregulated Bax and the active subunit of caspase-3, and downregulated Bcl-2 expression, as shown via Hoechst staining and flow cytometry analysis. Conclusion: Our findings suggest SMI enhances cisplatin-induced apoptosis through regulation of Mfn2-dependent mitochondrial dynamics in cisplatin-resistant lung adenocarcinoma cells.

Antitumor effects of dioscin in A431 cells via adjusting ATM/p53-mediated cell apoptosis, DNA damage and migration.

Skin cancer is the deadliest type of malignant disease and causes primary mortality worldwide. Dioscin, which exists in medicinal plants, has potent anticancer effects. However, its effects on skin cancer remain unknown. In the present study, the activity and mechanism of dioscin on the human skin cancer A431 cell line were investigated, MTT, colony formation, Transwell, wound-healing, TUNEL, Comet, immunofluorescence and western blot assays were used to assess the effects of dioscin on A431 cells. The results of MTT, colony formation, Transwell and wound-healing assays revealed that dioscin suppressed proliferation, colony formation and invasion of the cancer cells. TUNEL and comet assays demonstrated that dioscin exhibited significant effects on cell apoptosis and DNA damage. Investigations into the mechanism revealed that the expression levels of phosphorylated Ataxia telangiectasia-mutated (ATM) were considerably activated by dioscin, which significantly upregulated the expression levels of p53 to activate mitochondrial apoptosis signaling. Furthermore, the expression levels of BAX, cleaved caspase-3/9 and cleaved poly (ADP-ribose) polymerase were upregulated, and the expression levels of BCL-2 were downregulated by dioscin. Additionally, dioscin markedly downregulated the expression levels of matrix metalloproteinase 2 (MMP2), MMP9, RHO and cdc42, which are all associated with tumor invasion. In addition, p53-small interfering RNA transfection experiments indicated that dioscin exhibited excellent activity against skin cancer in vitro by decreasing p53 expression. Overall, the present results suggested that dioscin inhibited skin cancer cell proliferation via adjusting ATM/p53-mediated cell apoptosis, migration and DNA damage, which should be considered as a potential option for future treatments of skin cancer.

Shenmai Injection Supresses Glycolysis and Enhances Cisplatin Cytotoxicity in Cisplatin-Resistant A549/DDP Cells via the AKT-mTOR-c-Myc Signaling Pathway.

Tumor cells, especially drug-resistant cells, predominately support growth by glycolysis even under the condition of adequate oxygen, which is known as the Warburg effect. Glucose metabolism reprogramming is one of the main factors causing tumor resistance. Previous studies on Shenmai injection (SMI), a Chinese herbal medicine, have shown enhanced efficacy in the treatment of tumors in combination with chemotherapy drugs, but the mechanism is not clear. In this study, we investigated the effect of SMI combined with cisplatin on cisplatin-resistant lung adenocarcinoma A549/DDP cells. Our results showed that cisplatin-resistant A549/DDP cells exhibited increased glucose consumption, lactate production, and expression levels of key glycolytic enzymes, including hexokinase 2 (HK2), pyruvate kinase M1/2 (PKM1/2), pyruvate kinase M2 (PKM2), glucose transporter 1 (GLUT1), and lactate dehydrogenase A (LDHA), compared with cisplatin-sensitive A549 cells. SMI combined with cisplatin in A549/DDP cells, led to significantly lower expression levels of key glycolytic enzymes, such as HK2, PKM1/2, GLUT1, and pyruvate dehydrogenase (PDH). In addition, we found that the combination of SMI and cisplatin could inhibit cell proliferation and promote apoptosis by reducing the expression levels of p-Akt, p-mTOR, and c-Myc, and then, it reduced the glycolysis level. These results suggest that SMI enhances the antitumor effect of cisplatin via glucose metabolism reprogramming. Therefore, the combination of SMI and cisplatin may be a potential therapeutic strategy to treat cisplatin-resistant nonsmall cell lung cancer.

Shenqi Fuzheng Injection Reverses Cisplatin Resistance through Mitofusin-2-Mediated Cell Cycle Arrest and Apoptosis in A549/DDP Cells.

The goal of this evaluation was to examine the mechanisms of Shenqi Fuzheng injection (SFI), an extract made from the plants Radix Astragali and Radix Codonopsis, in the process of chemotherapy sensitivity in non-small-cell lung cancer (NSCLC) cells. We investigated the expression of mitofusin-2 (Mfn2), a mitochondrial GTPase that may be related to chemoresistance, and found that Mfn2 expression was lower in human cisplatin-resistant lung carcinoma A549/DDP cells than in cisplatin-susceptible A549 cells. Chemosensitivity to cisplatin was restored in A549/DDP cells following supplementation in conjunction with SFI treatment, the effect of which we evaluated via cell cycle, apoptosis, and cell signaling analysis. We found that the combined use of A549/DDP cells with SFI and cisplatin enhanced cell cycle arrested in the G2/M phase, which was accompanied by upregulation of p53 and p21 protein expression and induced mitochondrial apoptosis in conjunction with the upregulation of Bax and the downregulation of Bcl-2 protein expression. Moreover, cell cycle arrest and mitochondrial apoptosis coincided with the upregulation of Mfn2 expression, which, in turn, was related to the increased mitochondrial membrane permeabilization and elevated reactive oxygen species. In summary, our findings suggest that the effect of SFI in increasing chemotherapy sensitivity in cisplatin resistance of NSCLCs occurs through cell cycle arrest and the initiation of mitochondrial apoptosis involved in the upregulation of Mfn2 expression.

Identification and characterization of microRNAs in tree peony during chilling induced dormancy release by high-throughput sequencing.

Tree peony, one of the most valuable horticultural and medicinal plants in the world, has to go through winter to break dormancy. Growing studies from molecular aspects on dormancy release process have been reported, but inadequate study has been done on miRNA-guided regulation in tree peony. In this study, high-throughput sequencing was employed to identify and characterize miRNAs in three libraries (6 d, 18 d and 24 d chilling treatments). There were 7,122, 10,076 and 9,097 unique miRNA sequences belonging to 52, 87 and 68 miRNA families, respectively. A total of 32 conserved miRNAs and 17 putative novel miRNAs were identified during dormancy release. There were 771 unigenes as potential targets of 62 miRNA families. Total 112 known miRNAs were differentially expressed, of which 55 miRNAs were shared among three libraries and 28 miRNAs were only found in 18 d chilling duration library. The expression patterns of 15 conserved miRNAs were validated and classified into four types by RT-qPCR. Combining with our microarray data under same treatments, five miRNAs (miR156k, miR159a, miR167a, miR169a and miR172a) were inversely correlated to those of their target genes. Our results would provide new molecular basis about dormancy release in tree peony.

[Effects of Buzhong Yiqi decoction on expression of Bad, NF-κB, caspase-9, Survivin, and mTOR in nude mice with A549/DDP transplantation tumors].

This study was aimed to explore the effects of Buzhong Yiqi decoction on the expression levels of Bad, NF-κB, caspase-9, Survivin, and mTOR in nude mice with A549/DDP transplantation tumors.Sixty BALB/C mice were randomly divided into blank control group, tumor-bearing control group, cisplatin group and Buzhong Yiqi decoction of high, medium and low doses+cisplatin groups (hereinafter referred to as the high,medium and low combined groups). A549/DDP cells (concentration of 5×106 cells/mL)were cultured and inoculated in various groups, then the tumor-forming situations were observed. Corresponding treatment was given in all groups. Fourteen days later, immunohistochemistry and Real-time PCR methods were used to detect the expression levels of Bad, NF-κB, caspase-9, Survivin, mTOR protein and mRNA in tumors.Results showed that Buzhong Yiqi decoction combined with cisplatin could reduce the volume of transplanted tumors, and there was significant difference between medium combined group and high combined group(P<0.05). As compared with the tumor-bearing control group, the expression levels of Bad, NF-κB, Survivin and mTOR were significantly reduced in medium and high combined groups(P<0.05); the protein and mRNA expression levels of caspase-9 were gradually increased in medium combined and high combined groups(P<0.05), with statistical difference with tumor-bearing control group(P<0.05). There were statistical difference in mRNA expression of Bad, NF-κB and caspase-9 between medium combined group, high combined group and cisplatin group, low-combined group, tumor-bearing control group(P<0.05), but there was no statistical difference between cisplatin group, low-combined group, and tumor-bearing control group. In addition, there was no statistical difference between medium combined group and high combined group in protein and mRNA expression levels of various factors. Experimental results showed that Buzhong Yiqi decoction combined with cisplatin can inhibit the growth of A549/DDP transplanted tumors, and the mechanism may be associated with regulating Bad, NF-κB, caspase-9, Survivin, and mTOR levels as well as promoting apoptosis.

Costunolide and dehydrocostuslactone combination treatment inhibit breast cancer by inducing cell cycle arrest and apoptosis through c-Myc/p53 and AKT/14-3-3 pathway.

Our previous studies demonstrated that volatile oil from saussurea lappa root (VOSL), rich in two natural sesquiterpene lactones, costunolide (Cos) and dehydrocostuslactone (Dehy), exerts better anti-breast cancer efficacy and lower side effects than Cos or Dehy alone in vivo, however, their anti-cancer molecular mechanisms were still unknown. In this study, we investigated the underlying mechanisms of Cos and Dehy combination treatment (CD) on breast cancer cells through proteomics technology coupled with Western blot validation. Ingenuity Pathways Analysis (IPA) results based on the differentially expressed proteins revealed that both VOSL and CD affect the 14-3-3-mediated signaling, c-Myc mediated apoptosis signaling and protein kinase A (PKA) signaling. Western blot coupled with cell cycle and apoptosis analysis validated the results of proteomics analysis. Cell cycle arrest and apoptosis were induced in a dose-dependent manner, and the expressions of p53 and p-14-3-3 were significantly up-regulated, whereas the expressions of c-Myc, p-AKT, p-BID were significantly down-regulated, furthermore, the ratio of BAX/BCL-2 were significantly increased in breast cancer cells after CD and VOSL treatment. The findings indicated that VOSL and CD could induce breast cancer cell cycle arrest and apoptosis through c-Myc/p53 and AKT/14-3-3 signaling pathways and may be novel effective candidates for breast cancer treatment.

Volatile oil from Saussurea lappa exerts antitumor efficacy by inhibiting epithelial growth factor receptor tyrosine kinase-mediated signaling pathway in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) treatment remains lack of effective chemotherapeutic drugs, therefore, discovering novel anti-HCC drugs is a very attractive and urgent task. In this study, we reported VOSL (volatile oil from Saussurea lappa root) exhibits potent therapeutic effect on SMMC-7721 xenografts without obvious side effects. In the in vitro experiments, VOSL inhibited HCC cell proliferation by arresting cell cycle at S and G2/M phases, and induced HCC cell apoptosis by activating the Caspase3 pathway. VOSL also decreased the capability of HCC cell migration and invasion through MMP-9 depression. Moreover, mechanistic study indicated that VOSL can act as an epithelial growth factor receptor (EGFR) inhibitor to suppress EGFR activation and then to suppress its downstream MEK/P38 and PI3-K/Akt pathways. These results suggested that VOSL may be a novel anti-HCC drug candidate.

[Buzhong yiqi decoction containing serum reversed resistance of A549/DDP to cisplatin and its effect on the expression of survivin: an experimental research ].

OBJECTIVE: To analyze the reversal effect of Buzhong Yiqi Decoction (BYD) on multidrug resistance of human adenocarcinoma of lung cell line A549/DDP, and to study its effect on the expression of survivin by using serum pharmacological methods in vitro. Methods Totally 24 SD rats were divided into the high, medium and low dose groups, and the blank control group by randomized controlled method. The high dose BYD containing serum (1. 134 g/mL, 2 mL), the middle dose BYD containing serum (0.576 g/mL, 2 mL), and the low dose BYD containing serum (0.284 g/mL, 2 mL) were prepared. The inhibitory effects of different dose and concentrations BYD on the proliferation of A549 and A549/DDP cells were detected by MTT assay, and the drug resistance reversal fold was calculated. The expression of Survivin in the two cell strains were detected respectively by immunohistochemical assay, Western blot, and immunofluorescence method. RESULTS: BYD containing serum showed obvious inhibitory effect on the growth of A549 and 549/DDP. The inhibition rates of 10% dose groups were higher than those of 5% dose groups. Besides, it gradually increased along with increased concentrations. Compared with 10% blank control group, the inhibition rate increased in 10% middle and low dose groups (P <0.05). After acted with 10% middle dose BYD containing serum, IC50, of A549 and A549/DDP were both reduced (P <0.05), reversal fold (RF) both increased. Its reversal ratio on A549/DDP cells was 2. 46, decreasing the resistance of A549/DDP to DDP. Compared with A549 in the same group, the expression of Survivin was detected to decrease by immunocytochemical assay, Western blot, and immunofluorescence method (P<0.05). Compared with 10% blank control group, the inhibition rate decreased in 10% middle dose group (P <0. 05). CONCLUSIONS: 10% middle dose BYD containing serum could significantly inhibit the apoptosis of A549 and A549/DDP. Besides, it could moderately reverse the multidrug resistance of A549/DDP cells to DDP possibly through reducing the intracellular expression of Survivin and enhancing the sensitivity 549/DDP to chemotherapeutics.

[Effect of Buzhong Yiqi decoction on PI3K and AKT in spleen, stomach and lung of nude mice with lung adenocarcinoma transplantation tumor].

OBJECTIVE: To explore the effect of Buzhong Yiqi decoction on PI3K/AKT signaling pathway in spleen, stomach and lung of nude mice with lung adenocarcinoma transplantation tumor. METHOD: Totally 60 nude mice were randomly divided into the blank control group, the tumor-bearing control group, the cisplatin group, the low-dose Buzhong Yiqi decoction group, the middle-dose Buzhong Yiqi decoction group and the high-dose Buzhong Yiqi decoction group. After the corresponding interventions, efforts were made to measure the transplanted tumor volume and calculate the tumor inhibiting rate. The immunohistochemical method and real time PCR were used to detect the expression of PI3K and AKT level in nude mice spleen, stomach and lung. RESULT: Buzhong Yiqi decoction of different concentrations combined with cisplatin could inhibit the growth of the transplanted tumor, with the strongest inhibitory effect in the middle-dose Buzhong Yiqi decoction group and the high-dose Buzhong Yiqi decoction group. All of the expressions of PI3K and AKT protein and gene in the spleen, stomach and lung increased, with the most significant increase in the tumor-bearing group. Along with the increase of the concentration of cisplatin and Buzhong Yiqi decoction, the expressions of PI3K and AKT gradually reduced. Compared with the tumor-bearing control group, there were statistical differences in spleen and stomach tissues (P < 0.05). Compared with the cisplatin group, the middle-dose Buzhong Yiqi decoction group and the high-dose Buzhong Yiqi decoction group showed statistical differences (P < 0.05), but without statistical difference compared with the blank control group. CONCLUSION: Among nude mice with lung adenocarcinoma transplantation tumor, the PI3K and AKT protein and gene expressions in spleen, stomach and lung tissues increased, which might indicated the effect of cisplatin and Buzhong Yiqi decoction in reducing PI3K and AKT expressions and the relations between the reduction degree and the concentrations of Buzhong Yiqi decoction. Cisplatin combined with Buzhong Yiqi decoction could decrease the PI3K and AKT protein and gene expression in spleen, stomach and lung, and make the pathway closer to normal, so as to protect the functions of spleen, stomach and lung, there may be target spots of Buzhong Yiqi decoction in PI3K/AKT signal pathway.

Transcriptome analysis of tree peony during chilling requirement fulfillment: assembling, annotation and markers discovering.

Tree peony (Paeonia suffruticosa Andrews) is a well-known horticultural and medicinal plant. The flower buds must go through a period of endo-dormancy before bud sprouting in winter, but very little information concerned with dormancy release is available. We obtained 625,342 sequencing reads with massive parallel pyrosequencing on the Roche 454 GS FLX platform (mean length: 358.1bp). De novo assemblies yielded 23,652 contigs and singletons. 15,284 contigs longer than 300bp were further annotated, among them 12,345 ESTs showed significant similarity with sequences present in public databases (with an E-value <1e-10). 484 putative transcription factors were obtained. In addition, 2253 potential Simple Sequence Repeats (SSR) loci were identified in the 454-ESTs. Total 149 pairs of primers were designed, and 121 pairs were amplified successfully in initial screening. In addition, 73 pairs of primers displayed polymorphism. This sequence collection provides a significant resource for gene discovery during endo-dormancy of tree peony.