Daurisoline inhibits proliferation, induces apoptosis, and enhances TRAIL sensitivity of breast cancer cells by upregulating DR5.

Daurisoline (DS) is an isoquinoline alkaloid that exerts anticancer activities in various cancer cells. However, the underlying mechanisms through which DS affects the survival of breast cancer cells remain poorly understood. Therefore, the present study was undertaken to investigate the potential anticancer effect of DS on breast cancer cells and reveal the mechanism underlying the enhanced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by DS. Cell counting kit-8 (CCK-8) and 5-ethynyl-2-deoxyuridine (EdU) assay were used to evaluate the ability of cell proliferation. Flow cytometry was selected to examine the cell cycle distribution. TUNEL assay was used to detect the cell apoptosis. The protein expression was measured by Western blot analysis. DS was found to reduce the cell viability and suppress the proliferation of MCF-7 and MDA-MB-231 cells by causing G1 phase cell cycle arrest. DS could trigger apoptosis by promoting the cleavage of caspase-8 and PARP. The phosphorylation of ERK, JNK, and p38MAPK was upregulated clearly following DS treatment. Notably, SP600125 (JNK inhibitor) pretreatment significantly abrogated DS-induced PARP cleavage. DS inactivated Akt/mTOR and Wnt/ß-catenin signaling pathway and upregulated the expression of ER stress-related proteins. Additionally, DS amplified TRAIL-caused viability reduction and apoptosis in breast cancer cells. Mechanismly, DS upregulated the protein level of DR4 and DR5, and knockdown of DR5 attenuated the cotreatment-induced cleavage of PARP. Inhibition of JNK could block DS-induced upregulation of DR5. This study provides valuable insights into the mechanisms of DS inhibiting cell proliferation, triggering apoptosis, and enhancing TRAIL sensitivity of breast cancer cells.

6-Methoxydihydrosanguinarine exhibits cytotoxicity and sensitizes TRAIL-induced apoptosis of hepatocellular carcinoma cells through ROS-mediated upregulation of DR5.

6-methoxydihydrosanguinarine (6-MS), a natural benzophenanthridine alkaloid extracted from Macleaya cordata (Willd.) R. Br, has shown to trigger apoptotic cell death in cancer cells. However, the exact mechanisms involved have not yet been clarified. The current study reveals the underlying mechanisms of 6-MS-induced cytotoxicity in hepatocellular carcinoma (HCC) cells and investigates whether 6-MS sensitizes TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis. 6-MS was shown to suppress cell proliferation and trigger cell cycle arrest, DNA damage, and apoptosis in HCC cells. Mechanisms analysis indicated that 6-MS promoted reactive oxygen species (ROS) generation, JNK activation, and inhibits EGFR/Akt signaling pathway. DNA damage and apoptosis induced by 6-MS were reversed following N-acetyl-l-cysteine (NAC) treatment. The enhancement of PARP cleavage caused by 6-MS was abrogated by pretreatment with JNK inhibitor SP600125. Furthermore, 6-MS enhanced TRAIL-mediated HCC cells apoptosis by upregulating the cell surface receptor DR5 expression. Pretreatment with NAC attenuated 6-MS-upregulated DR5 protein expression and alleviated cotreatment-induced viability reduction, cleavage of caspase-8, caspase-9, and PARP. Overall, our results suggest that 6-MS exerts cytotoxicity by modulating ROS generation, EGFR/Akt signaling, and JNK activation in HCC cells. 6-MS potentiates TRAIL-induced apoptosis through upregulation of DR5 via ROS generation. The combination of 6-MS with TRAIL may be a promising strategy and warrants further investigation.

Acetylshikonin induces autophagy-dependent apoptosis through the key LKB1-AMPK and PI3K/Akt-regulated mTOR signalling pathways in HL-60 cells.

Acetylshikonin (ASK) is a natural naphthoquinone derivative of traditional Chinese medicine Lithospermum erythrorhyzon. It has been reported that ASK has bactericidal, anti-inflammatory and antitumour effects. However, whether ASK induces apoptosis and autophagy in acute myeloid leukaemia (AML) cells and the underlying mechanism are still unclear. Here, we explored the roles of apoptosis and autophagy in ASK-induced cell death and the potential molecular mechanisms in human AML HL-60 cells. The results demonstrated that ASK remarkably inhibited the cell proliferation, viability and induced apoptosis in HL-60 cells through the mitochondrial pathway, and ASK promoted cell cycle arrest in the S-phase. In addition, the increased formation of autophagosomes, the turnover from light chain 3B (LC3B) I to LC3B II and decrease of P62 suggested the induction of autophagy by ASK. Furthermore, ASK significantly decreased PI3K, phospho-Akt and p-p70S6K expression, while enhanced phospho-AMP-activated protein kinase (AMPK) and phospho-liver kinase B1(LKB1) expression. The suppression of ASK-induced the conversion from LC3B I to LC3B II caused by the application of inhibitors of AMPK (compound C) demonstrated that ASK-induced autophagy depends on the LKB1/AMPK pathway. These data suggested that the autophagy induced by ASK were dependent on the activation of LKB1/AMPK signalling and suppression of PI3K/Akt/mTOR pathways. The cleavage of the apoptosis-related markers caspase-3 and caspase-9 and the activity of caspase-3 induced by ASK were markedly reduced by inhibitor of AMPK (compound C), an autophagy inhibitor 3-methyladenine (3-MA) and another autophagy inhibitor chloroquine (CQ). Taken together, our data reveal that ASK-induced HL-60 cell apoptosis is dependent on the activation of autophagy via the LKB1/AMPK and PI3K/Akt-regulated mTOR signalling pathways.

CUDC-907 enhances TRAIL-induced apoptosis through upregulation of DR5 in breast cancer cells.

CUDC-907 is a novel dual-acting inhibitor of phosphoinositide 3-kinase (PI3K) and histone deacetylase (HDAC). In this study, we aimed to explore the anticancer effects of CUDC-907 on human breast cancer cells. Our results showed that CUDC-907 effectively inhibited breast cancer cell proliferation. Flow cytometry analysis revealed that CUDC-907 induced cell cycle arrest and apoptosis in breast cancer cells. The combined treatment of CUDC-907 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resulted in a marked increase in apoptosis and cleavage of caspase-8, -9 and poly (ADP-ribose) polymerase (PARP) in breast cancer cells. CUDC-907 enhanced expressions of death receptor 5 (DR5), reduced the levels of anti-apoptotic molecules XIAP, Bcl-2 and Bcl-xL. Knockdown of DR5 abrogated apoptosis induced by the combination of CUDC-907 and TRAIL in breast cancer cells. CUDC-907 increased the phosphorylation of JNK and p38 MAPK. JNK inhibitor pretreatment attenuated CUDC-907-induced upregulation of DR5. In summary, CUDC-907 shows potent cytotoxicity against breast cancer cells and facilitates TRAIL-mediated apoptosis through DR5 upregulation. The combination of CUDC-907 and TRAIL may be a promising therapeutic approach in the treatment of breast cancer.

Acetoacetate extract of pleione bulbocodioides (franch. ) rolfe induces apoptosis of human leukemia thp-1cells through a mitochondria-regulated intrinsic apoptotic pathway / Extrato de acetoacetato de Pleione bulbocodioides (Franch. ) Rolfe induz a apoptose de células THP-1 de leucemia humana através de uma via apoptótica intrínseca regulada por mitocôndrias

The tubers of three orchidaceous plants, includingPleione bulbocodioides (Franch.) Rolfe, have been used as 'Shan-Ci-Gu' in traditional Chinese medicine for the treatment of bacterial infections and cancers for thousands of years. In this study, the effects of an acetoacetate (EtOAc) extract of P. bulbocodioides on the cell viability and apoptosis of THP-1 (human acute monocytic leukemia cell line) cells and its interaction with possible apoptotic pathways were investigated. THP-1 cells were treated with the EtOAc extract of P. bulbocodioides at different concentrations. The results showed that THP-1 cell viability was significantly inhibited by the EtOAc extract ofP. bulbocodioides with an IC50 of 51.37±2.68 µ g/ mL at 24 h. The examination of cytotoxic effects on healthy cells showed that the EtOAc extract of P. bulbocodioidesdid not show any effect on healthy Vero cells. Selectivity indexes were greater than 15.57, suggesting that the EtOAc extract of P. bulbocodioides had selective toxicity against THP-1 cells. The results of annexin V-FITC/PI and DAPI staining showed that the EtOAc extract of P. bulbocodioides induced cell apoptosis in a dose-dependent manner. The apoptotic rate was increased in the treatment groups compared with that in the control group (P<0.05). The distribution of cells in the G2 phase of the cell cycle increased along with typical cell apoptosis-induced morphological changes. The levels of the pro-apoptotic proteins Bax, cleaved PARP and cleaved caspase-3 increased with increasing concentration of acetoacetate extract of P. bulbocodioides, while the anti-apoptosis protein Bcl-2 was downregulated. Cyt c and AIF, which are characteristic proteins of the mitochondria-regulated intrinsic apoptosis pathway, also increased in the cytosol with increasing concentrations of the EtOAc extract of P. bulbocodioides. These results showed that the EtOAc extract of P. bulbocodioidessignificantly inhibits cell viability and induces cell apoptosis in the human leukemia cell line THP-1 through a mitochondria-regulated intrinsic apoptotic pathway

Os tubérculos de três plantas orquidáceas, incluindo Pleione bulbocodioides (Franch.) Rolfe, têm sido usados como "Shan-Ci-Gu" na medicina tradicional chinesa para o tratamento de infecções bacterianas e cânceres por milhares de anos. Neste estudo, os efeitos de um extrato de acetoacetato (EtOAc) de P. bulbocodioides na viabilidade celular e apoptose de células THP-1 (linhagem celular de leucemia monocítica aguda humana) e sua interação com possíveis vias apoptóticas foram investigados. As células THP-1 foram tratadas com o extrato EtOAc de P. bulbocodioides em diferentes concentrações. Os resultados mostraram que a viabilidade das células THP-1 foi significativamente inibida pelo extrato EtOAc de P. bulbocodioides com IC50 de 51,37 ± 2,68 µ g/mL às 24 h. O exame dos efeitos citotóxicos em células saudáveis mostrou que oextrato de EtOAc de P. bulbocodioides não mostrou nenhum efeito sobre células Vero saudáveis. Os índices de seletividade foram maiores que 15,57, sugerindo que o extrato de EtOAc de P. bulbocodioides teve toxicidade seletiva contra as células THP-1. Os resultados da coloração da anexina V-FITC/PI e DAPI mostraram que o extrato de EtOAc de P. bulbocodioides induziu a apoptose celular de maneira dose-dependente. A taxa de apoptose foi aumentada nos grupos de tratamento em comparação com o grupo controle (P <0,05). A distribuição de células na fase G2 do ciclo celular aumentou juntamente com alterações morfológicas típicas induzidas pela apoptose celular. Os níveis das proteínas pró-apoptóticas Bax, PARP clivada e caspase-3 clivada aumentaram com o aumento da concentração do extrato acetoacetato de P. bulbocodioides, enquanto a proteína anti-apoptose Bcl-2 foi regulada negativamente. Cyt c e AIF, que são proteínas características da via de apoptose intrínseca regulada por mitocôndrias, também aumentaram no citosol com concentrações crescentes do extrato de EtOAc de P. bulbocodioides. Estes resultados mostraram que o extrato de EtOAc de P. bulbocodioides inibe significativamente a viabilidade celular e induz a apoptose na linha celular de leucemia humana THP-1 através de uma via apoptótica intrínseca regulada por mitocôndrias.

The endophytic fungi of Salvia miltiorrhiza Bge.f. alba are a potential source of natural antioxidants.

BACKGROUND: Salvia miltiorrhiza Bge. f. alba is a traditional Chinese herbal drug with special pharmacological effect on thromboangiitis obliterans. However, the nature source of S.miltiorrhiza Bge.f.alba is now in short supply because of the over-collection of the wild plant. To better utilize this resource, the diversity and antioxidant activity of endophytic fungi isolated from S. miltiorrhiza Bge. f. alba were investigated. RESULTS: A total of 14 endophytic fungi were isolated from different parts of S. miltiorrhiza Bge.f.alba. Based on morphological and molecular identification, the endophytic fungi isolated were classified into four genera (Alternaria sp., Fusarium sp., Schizophyllum sp. and Trametes sp.). These fungal extracts were prepared using ethanol and evaluated for their phytochemical compounds and antioxidant activity. Alternaria alternata SaF-2 and Fusarium proliferatum SaR-2 are of particular interest because they yielded all of nine phytochemicals including saponins, phenol, flavonoids, cardiac glycosides, steroids, tannins, alkaloids, anthroquinone and terpenoids. F. proliferatum SaR-2 and A. alternata SaF-2 also exhibited stronger antioxidant activities by FRAP and DPPH method, having the higher levels of phenol and flavonoid than those of plant root. The total amount of phenol and flavonoid quantified were of 21.75, 20.53 gallic acid equivalent per gram and 8.27 and 7.36 µg/mg of quercetin equivalent respectively. These two endophytic fungi (SaR-2 and SaF-2) were found to have comparable scavenging abilities on both FRAP (1682.21 and 1659.05 µmol/mg, respectively) and DPPH-free radicals (90.14% and 83.25%, respectively, at 0.1 mg/mL). This is the first report about isolation of endophytic fungi from S. miltiorrhiza Bge.f.alba and their antioxidant activities. CONCLUSIONS: These results indicate that the endophytic fungi associated with S. miltiorrhiza Bge.f. alba can be a potential source of novel natural antioxidants.

[Construction of plant expression vectors with PMI gene as selection marker and their utilization in transformation of Salvia miltiorrhiza f. alba].

OBJECTIVE: To construct plant expression pCAMBIA1301-PMI by substituting PMI for hygromycin resistance gene in pCAMBIA1301 and obtain transgenic Salvia miltiorrhiza f. alba using PMI-mannose selection system. METHOD: The 6-phosphomannose isomerase gene (PMI) of Escherichia coli was amplified by PCR. Sequence analysis showed that it shared 100% amino acids identities with the sequences of PMI genes isolates reported in the NCBI. Based on pCAMBIA1305, the plant expression pCAMBIA1305-PMI was constructed successfully by substituting PMI for hygromycin resistance gene in pCAMBIA1305. pCAMBIA1305-PMI was transformed into Agrobacterium tumefaciens LBA4404, and then the leaves of S. miltiorrhiza f. alba were inoculated in LBA4404 with pCAMBIA1305-PMI. RESULT: Plant expression pCAMBIA1301-PMI was successfully constructed and the leaves of S. miltiorrhiza f. alba inoculated in LBA4404 with pCAMBIA1305-PMI were selected on medium supplemented with a combination of 20 g x L(-1) mannose and 10 g x L(-1) sucrose as a carbon source. The transformation efficiency rate was 23.7%. CONCLUSION: Genetic transformation was confirmed by PCR, indicating that a new method for obtaining transgenic S. miltiorrhiza f. alba plants was developed using PMI-mannose selection system.

[Study on effective constituents extracted from fibrous roots of Salvia miltiorrhiza with degrading multi-enzymes from taishan Ganoderma lucidum].

OBJECTIVE: To study the application of degrading multi-enzymes from Ganoderma lucidum in extracting effective constituents from fibrous roots of Salvia miltiorrhiza. METHOD: Effective constituents were extracted from fibrous roots by degrading multi-enzymes of wood fiber. The enzymatic parameters were optimized by the orthogonal design. RESULT: The extraction efficiencies of total tanshinones and total salvianolic acids in the extracts of fibrous roots of S. miltiorrhiza was obtained using optimum enzymolysis process reached 11.923%, 12.465%, respectively, which were 62.794%, 56.086% more than that by conventional non-enzymatic hydrolysis. CONCLUSION: Degrading multi-enzymes of wood fiber can be used to fully extract effective constituents from fibrous roots of S. miltiorrhiza, which provides a new approach for recycling wastes of traditional Chinese medicines.

Antitumor activity of decoy oligodeoxynucleotides targeted to NF-kappaB in vitro and in vivo.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB), a transcription factor, is abundantly expressed in many tumors and regulates many tumor-relative genes such as c-myc and caspase-8, which NF-kappaB-mediated genes activation serves as an anti-tumor pathway by regulating expression of these tumor-relative genes. Given the important roles of these genes in tumor control, the present study was to test the hypothesis that NF-kappaB decoy ODNs transfected into tumor cells in vitro and in vivo might affect growth and apoptosis. METHODS: First, NF-kappaB decoy oligodeoxynucleotides were designed according to the NF-kappaB elements in the promoter region of c-myc gene. Then, supression effects of decoy ODNs on proliferation of five carcinoma cell lines were assessed by MTT assay. Apoptosis of carcinoma cells was determined by chromosome DNA ladder and flow cytometric analysis (FCM). Thirdly, suppression effects of NF-kappaB decoy ODNs on proliferation of carcinoma in vivo were investigated in nude mice. To confirm mechanisms of action, a pGL3-C-MYC luciferase expression vector containing a fragment of the c-myc promoter was constructed and co-transfected with NF-kappaB decoy ODNs into SKOV-3 cells by lipofectamine TM2000. Expression levels of endogenous c-myc and caspase-8 genes were assessed by northern blotting. Lastly, nuclear extracts were prepared from SKOV-3 cells and DNA-protein interactions were examined by electrophoretic mobility shift assay (EMSA). RESULTS: Treatment of cancer cell lines with NF-kappaB decoy ODNs resulted in strong suppression of proliferation, especial of the SKOV-3 ovarian cancer cell line in vitro and in vivo. Induction of apoptosis of SKOV-3 was observed in DNA gel electrophoresis and FCM. Activity of luciferase was significantly reduced in the NF-kB decoy-transfected cells, but not in cells transfected with a control decoy. Furthermore, we found that transcripts of endogenous c-myc gene were reduced, while caspase-8 transcripts were induced. EMSA demonstrated specific binding of the NF-kappaB decoy to NF-kappaB protein. CONCLUSIONS: These findings indicatd that NF-kappaB activation plays an important role in proliferation in many cancers, esspecially ovarian carcinomas. Inhibitors of NF-kappaB may thus offer promise as a therapeutic approach for the treatment of tumors via manipulating expression of desired target genes. NF-kappaB decoy ODNs may allow development of therapeutic and investigative tools for human malignancies.

Nucleotide variation in the NCED3 region of Arabidopsis thaliana and its association study with abscisic acid content under drought stress.

Drought tolerance is a comprehensive quantitative trait that is being understood further at the molecular genetic level. Abscisic acid (ABA) is the main drought-induced hormone that regulates the expression of many genes related to drought responses. 9-cis-epoxycarotenoid dioxygenase (NCED3) is thought to be a key enzyme in ABA biosynthesis. In this paper, we measured the ABA content increase under drought stress, and sequenced and compared the sequence of AtNCED3 among 22 Arabidopsis thaliana accessions. The results showed that the fold of ABA content increase under drought stress was highly variable among these accessions. High density single nucleotide polymorphism (SNP) and insertion/deletion (indel) were found in the AtNCED3 region, on average one SNP per 87.4 bp and one indel per 502 bp. Nucleotide diversity was significantly lower in the coding region than that in non-coding regions. The results of an association study with anova analysis suggested that the 274th site (P<-->S) and the 327th site (P<-->R) amino acid variations might be the cause of ABA content increase of 163av accession under drought stress.

Role of nitric oxide dependence on nitric oxide synthase-like activity in the water stress signaling of maize seedling.

Nitric oxide (NO) has been known as an important signal in plant antioxidative defense but its production and roles in water stress are less known. The present study investigated whether NO dependence on a NO synthase-like (NOS) activity is involved in the signaling of drought-induced protective responses in maize seedlings.NOS activity, rate of NO release and drought responses were analyzed when NO donor sodium nitroprusside (SNP), NO scavenger c-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and NOS inhibitor L-NAME (NG-nitro-L-arginine methyl ester) were applied to both detached maize leaves and whole plants. Both NOS activity and the rate of NO release increased substantially under dehydration stress. The high NOS activity induced by c-PTIO as NO scavenger and NO accumulation inhibited by NOS inhibitor L-NAME in dehydration-treated maize seedlings indicated that most NO production under water deficit stress may be generated from NOS-like activity. After dehydration stress for 3 h, detached maize leaves pretreated with NO donor SNP maintained more water content than that of control leaves pretreated with water. This result was consistent with the decrease in the transpiration rate of SNP-treated leaves subjected to drought treatment for 3 h. Membrane permeability, a cell injury index, was lower in SNP-treated maize leaves under dehydration stress for 4 h when compared with the control leaves. Also, superoxide dismutase (SOD) activity of SNP combined drought treatment maize leaves was higher than that of drought treatment alone, indicating that exogenous NO treatment alleviated the water loss and oxidative damage of maize leaves under water deficit stress. When c-PTIO as a specific NO scavenger was applied, the effects of applied SNP were overridden. Treatment with L-NAME on leaves also led to higher membrane permeability, higher transpiration rate and lower SOD activities than those of control leaves, indicating that NOS-like activity was involved in the antioxidative defense under water stress. These results suggested that NO dependence on NOS-like activity serves as a signaling component in the induction of protective responses and is associated with drought tolerance in maize seedlings.

[Exogenous nitric oxide accelerates soluble sugar, proline and secondary metabolite synthesis in Ginkgo biloba under drought stress].

The effects of exogenous nitric oxide donor sodium nitroprusside (SNP) on substance metabolism of Ginkgo biloba leaves under drought stress were studied. The results showed that 250 micromol/L SNP (Fig.2) treatment under 35% relative soil water content (RSWC) stress (Fig.1) raised remarkably soluble sugar content (Fig.3), proline content (Fig.4), phenylalanine ammonia lyase (PAL) activity (Fig.5), flavonoids (Fig.6) and ginkgolides content (Fig.7) of G. biloba leaves. Hemoglobin, used as NO scavenger, counteracted the effects of SNP in raising the soluble sugar (Fig.3), proline (Fig.4), flavonoid (Fig.6), ginkgolide content (Fig.7) and PAL activities (Fig.5), which indicates that the effects of sodium nitroprusside were through the nitric oxide released from sodium nitroprusside. We propose from these results that the roles of flavonoids and ginkgolides are the same as those of soluble sugars and proline under drought stress. NO may alleviate the damage caused by drought stress through raising soluble sugar, proline, flavonoid and ginkgolide content.

[ATHK1 gene regulates signal transduction of osmotic stress in Arabidopsis thaliana].

Differences in physiology and gene expression between ATHK1 knock-out mutant caused by T-DNA insertion and wild type (WT) of WS accession of Arabidopsis thaliana were analysed. Water loss ratio of detached leaf of ATHK1-mutant was obviously higher than that of WT. After being treated with 30% PEG-6000, ion leakage ratio of cell membrane in wild type leaves was 50% higher than that before PEG treatment, while in mutant leaves it increased 80%. The wilted phenotype of ATHK1-mutant after PEG treatment for 48 h was higher than that of WT. All these results showed that ATHK1-mutant was more sensitive to osmotic stress compared to WT and ATHK1 involved in osmotic stress adaptation. Differential-Display Reverse Transcription-PCR (DDRT-PCR) analysis was carried out to investigate the difference of gene expression between ATHK1-mutant and WT. Nine differential cDNA fragments involved in stress adaptation were identified, including the MAPKKK18 and serine/threonine protein kinase genes. These fragments were up-regulated by PEG treatment in WT, but not in ATHK1-mutant. These results indicate that ATHK1 plays an important role up-stream from MAPK in the osmotic stress signal transduction pathway. ATHK1 may be working as a plant osmosensor.

[Nucleotide polymorphism in the drought induced transcription factor CBF4 region of Arabidopsis thaliana and its molecular evolution analyses].

Intraspecific nucleotide polymorphism in the drought induced transcription factor CBF4 region of Arabidopsis thaliana was analyzed with 17 core accessions growing in different ecoclimate. High density of single nucleotide polymorphism (SNP) and insertion/deletion (Indel) were found, on average 1 SNP per 35.8 bp and 1 Indel per 143 bp. Nucleotide polymorphism in non-coding region was three times higher than that in coding region. In coding region of CBF4, SNP frequency is one SNP per 96.4 bp, one nonsynonymous mutation was detected from 25 av, 203 av and 244 av accessions, which is the 205th site amino acid variation: gly <--> val caused by the 1034th site (corresponding to 19,696 site nucleotide of GenBank No. AB015478 as 1) nucleotide variation: G <--> T. Statistical result of nucleotide diversity showed that linkage disequilibrium (LD) existed in large-scale region of CBF4 and recombination event was also detected in 5' non-coding region. Identical to the results of other genes of Arabidopsis, different regions of the gene were seemingly under different selective pressures. Balancing selection resulted in high nucleotide diversity in 3' non-coding region, and the neutral mutation hypothesis can explain the DNA polymorphism in coding region, whereas, nature positive selection in the population affected nucleotide variation in 5' non-coding region of gene.