Xueting Cai

Luteolin induced G2 phase cell cycle arrest and apoptosis on non-small cell lung cancer cells

In this study, we investigated the underlying molecular mechanism for the potent cell cycle inhibition and pro-apoptotic effect of luteolin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-chromenone) on human non-small-cell lung carcinoma cell line A549. MTT assay showed that luteolin had obvious cytotoxicity on A549 with IC(50) of 40.2 μM at 48 h. Pro-apoptotic effect of luteolin on A549 cells was demonstrated by Hoechst 33258 staining assay and annexin V-FITC/PI double staining analysis. A great quantity of apoptotic cells and increasing G2 phase cells were observed by flow cytometry. Western blotting assay revealed that luteolin activated JNK, increased Bax, promoted procaspase-9 cleavage and activated caspase-3 at last. Assay using TNFα, an active agent of NF-κB, showed that pretreatment of A549 cells with luteolin could inhibit TNFα induced trans-nuclear of NF-κB. In summary, luteolin displayed a significant cytotoxic effect through cell cycle arrest and apoptosis induction in A549 cells. Pro-apoptotic effect was implemented via activating JNK and inhibiting translocation of NF-κB (p65). These results suggested that luteolin might have therapeutic potential against NSCLC.

Analgesic‐antitumor peptide inhibits proliferation and migration of SHG‐44 human malignant glioma cells

Malignant gliomas, the most common subtype of primary brain tumors, are characterized by high proliferation, great invasion, and neurological destruction and considered to be the deadliest of human cancers. Analgesic‐antitumor peptide (AGAP), one of scorpion toxic polypeptides, has been shown to have antitumor activity. Here, we show that recombinant AGAP (rAGAP) not only inhibits the proliferation of gliomas cell SHG‐44 and rat glioma cell C6, but also suppresses the migration of SHG‐44 cells during wound healing. To explain these phenomena, we find that rAGAP leads to cell cycle of SHG‐44 arrested in G1 phase accompanied by suppressing G1 cell cycle regulatory proteins CDK2, CDK6, and p‐RB by means of the down‐regulated protein expression of p‐AKT. Meanwhile, rAGAP significantly decreases the production of NF‐κB, BCL‐2, p‐p38, p‐c‐Jun, and p‐Erk1/2 and further suppresses the activation of VEGF and MMP‐9 in SHG‐44 cells. These findings suggest rAGAP inhibit proliferation and migration of SHG‐44 cells by arresting cell cycle and interfering p‐AKT, NF‐κB, BCL‐2, and MAPK signaling pathways. J. Cell. Biochem. 112: 2424–2434, 2011.

Immunological regulation of Chinese herb Guizhi Fuling Capsule on rat endometriosis model.

AIM OF THE STUDYnTo investigate the immunological regulation of Guizhi Fuling Capsule (GZFLC) on rat endometriosis.nnnMATERIALS AND METHODSnTwenty-seven rats, in which endometriotic implants were induced by transplanting autologous uterine tissue to the peritoneum, were randomly divided into three groups equally: (1) the GZFLC group of low dose (480 mg/kg/day); (2) the GZFLC group of high dose (1,920 mg/kg/day); and (3) the model group(saline solution). Another 10 rats were treated as sham operation group. After rats were treated for four weeks, we examined the alterations of implants volume, the percentage of CD4(+) T lympholeukocyte, the activity of NK cell and the expression of cytokines (MCP-1 and ICAM-1) on each group.nnnRESULTSnStatistical analysis showed that posttreatment volumes were significantly reduced compared with pretreatment in GZFLC groups, whereas there was no significant change in the model group. The percentage of CD4(+) T lympholeukocyte and the activity of NK cell in GZFLC groups significantly increased to the level of the sham group compared with the model. RT-PCR and immunohistochemistry showed that the endometria of the sham operation and treatment groups were similar on expression level of MCP-1 and ICAM-1.nnnCONCLUSIONSnGZFLC plays an important role in the regression of endometriotic implants by immunological regulation in the rat model.

Banxia xiexin decoction protects against dextran sulfate sodium-induced chronic ulcerative colitis in mice

ETHNOPHARMACOLOGICAL RELEVANCEnBanxia Xiexin decoction (BXD), one of a traditional Chinese medicine chronicled in Shang Han Lun, is commonly used to treat gastroenteritis, ulcerative colitis and diarrhea. In our study, we used current biomedical approaches to investigate the therapeutic efficacy of BXD and possible protective mechanism involved in inhibiting dextran sulfate sodium (DSS)-induced chronic ulcerative colitis model.nnnMATERIALS AND METHODSnChronic DSS colitis was induced in C57BL/6 male mice by three cycles of 5 days of 2% DSS in drinking water, alternating with 5 days of normal water, totaling 30 days. In BXD group, the mice were administered at a dose of 8.7g/kg BXD for 5 days before and during DSS treatment via oral gavage per day. Mice in vehicle group and DSS group were given orally the same volume of drinking water, instead. Body weight, stool characters and hematochezia were observed everyday. The colorectal tissues were used to detect levels of TNF-α, IL-4, IL-10, IL-1β, IL-17, IL-23 and MPO by ELISA or qRT-PCR. The expression of COX-2, 8-Oxoguanine and Nrf2 were examined by IHC, and p-p65 was examined by western blotting. ThOD and the content of MDA were measured according to kits respectively.nnnRESULTSnBXD significantly protected against DSS-induced chronic ulcerative colitis by amelioration of body weight loss, DAI and histology score. The level of TNF-α, IL-1β, IL-17, IL-23, COX-2 and p-p65 were decreased significantly, while the level of IL-10 improved with the treatment of BXD. MDA, MPO and 8-Oxoguanine were decreased, meanwhile SOD activity and Nrf2 expression were elevated significantly by BXD.nnnCONCLUSIONSnBXD possesses the potential of anti-inflammation and anti-oxidation to treat colitis. The protective mechanism of BXD may involve in inhibition of NF-κBp65 activation and increasement of Nrf2 expression in colorectums of mice.

Escin Sodium Induces Apoptosis of Human Acute Leukemia Jurkat T Cells

Escin sodium has been used in the clinic as an antioedematous, antiexudative and vasoprotective agent for many years and has shown excellent tolerability. However, little is known about its anticancer activity. This is a report for the first time that escin sodium exerts a cytotoxic effect on human acute leukemia Jurkat T cells via the induction of apoptosis rather than cell cycle arrest. Escin sodium activated the initiator caspase‐8, ‐9, and the effector caspase‐3, degraded poly (ADP‐ribose) polymerase (PARP) and attenuated the expression of Bcl‐2. In addition, escin sodium inhibited the growth of cancer cells in a selective manner with Jurkat cells most sensitive to it. Taken together, the data show that escin sodium possesses potent apoptogenic activity toward human acute leukemia Jurkat T cells. Copyright

Expression and Purification of an Antitumor-Analgesic Peptide from the Venom of Mesobuthus martensii Karsch by Small Ubiquitin-Related Modifier Fusion in Escherichia coli

To prevent protein aggregation, some proteins are usually expressed as fusion proteins from which target proteins can be released by proteolytic or chemical reagents. In this report, small ubiquitin‐related modifier (SUMO) linked with a hexa‐histidine tag was used as a fusion partner for the antitumor‐analgesic peptide from the venom of Buthus martensii (Karsch) scorpion (AGAP). The optimal expression level of the soluble fusion protein, SUMO‐AGAP, was up to 40% of the total cellular protein. The fusion protein was purified by Ni‐NTA affinity chromatography and cleaved by a SUMO‐specific protease (Ulp1) to obtain the recombinant AGAP (rAGAP), which was further purified by Ni‐NTA affinity chromatography. The purified final product was >95% pure by SDS‐PAGE stained with Coomassie brilliant blue R‐250. Mass spectroscopic analysis indicated the protein to be 7142.63 Dalton, which equaled the theoretically expected mass. N‐terminal sequencing of rAGAP showed the sequence corresponded to the native protein. MTT assay indicated the rAGAP could significantly inhibit the proliferation of Jurkat and Hut 78 T lymphoma cell lines. The further writhing experiment showed that the rAGAP had an intensive analgesic effect. The expression strategy presented in this study allows convenient high yield and easy purification of the rAGAP with native sequences.

Molecular cloning, expression, and bioactivity of dove B lymphocyte stimulator (doBAFF)

B cell activating factor (BAFF) belonging to the tumor necrosis factor (TNF) family is a novel member of the tumor necrosis factor ligand family and plays an important role in B lymphocyte maturation and survival. cDNA of dove B lymphocyte stimulator (doBAFF) was amplified from total RNA of dove spleen by RT-PCR (reverse transcription PCR). The open reading frame of doBAFF consists 867 bases encoding a protein of 288 amino acids. Sequence comparison indicated the amino acid sequence of doBAFF showed high identity to hBAFF (50.66%) and cBAFF (91.32%). The result of RT-PCR showed that doBAFF was highly expressed in the spleen and bursa of fabricius. To enhance the soluble expression of doBAFF in Escherichia coli, we fused the extracellular region of doBAFF gene with a small ubiquitin-related modifier gene (SUMO) by over-lap PCR. The resulting fused protein SUMO-sdoBAFF was highly expressed in DE3(BL21) with a molecular weight of 35kDa. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease, Ulp1. The sdoBAFF protein was further purified by Ni-NTA affinity chromatography. In vitro, the MTT assays indicated that the purified doBAFF as well as SUMO-sdoBAFF proteins were able to promote bursa lymphocyte survival in dose-dependent manner.

Gut microbiota drives the attenuation of dextran sulphate sodium-induced colitis by Huangqin decoction

The gut microbiota, including probiotics and pathogenic microorganisms, is involved in ulcerative colitis (UC) by regulating pathogenic microorganisms and the production of intestinal mucosal antibodies. Huangqin decoction (HQD), a traditional Chinese formula chronicled in the Shanghan lun, has been recognized as an effective drug for UC, owing to its anti-inflammatory and anti-oxidative properties. In the present study, we investigated whether HQD ameliorates dextran sulphate sodium (DSS)-induced colitis through alteration of the gut microbiota. We found that HQD significantly inhibited colitis, alleviating the loss of body weight, disease activity index, colon shortening, tissue injury, and inflammatory cytokine changes induced by DSS treatment. Principal component analysis and principal co-ordinate analysis showed an obvious difference among the groups, with increased diversity in the DSS and DSS+HQD groups. Linear discriminant analysis effect size was used to determine differences between the groups. The relative abundance of Lactococcus was higher in the DSS+HQD group than in the DSS group, whereas Desulfovibrio and Helicobacter were decreased. Furthermore, the protective effect of HQD was attenuated only in antibiotic-treated mice. In conclusion, our results suggest that HQD could ameliorate DSS-induced inflammation through alteration of the gut microbiota.

Protective effects of Huangqin Decoction against ulcerative colitis and associated cancer in mice

Individuals with ulcerative colitis (UC) are at a high risk for developing colorectal cancer (CRC). Huangqin Decoction (HQD), a traditional Chinese medicinal formula chronicled in the Shang Han Lun, is commonly used to treat gastrointestinal symptoms. However, experimental evidence for supporting the clinical practice is lacking. This study used modern biomedical approaches to investigate the protective/preventive effects of HQD in dextran sulfate sodium (DSS)-induced acute/chronic UC and azoxymethane (AOM)/DSS-induced CRC in mice. HQDs were prepared in 4 different ways: HQD-1 and HQD-2 were prepared in boiling water, whereas HQD-3 and HQD-4 were prepared in heated ethanol (70%). For HQD-1 and HQD-3, the 4 constituent herbs were processed together, whereas for HQD-2 and HQD4, these herbs were processed individually and then combined. The mice were administered 9.1 g/kg HQD via oral gavage daily. HQD-1 significantly inhibited DSS-induced acute UC, whereas HQD-3 and HQD-4 exhibited mild ameliorative effects; but HQD-2 had no protective effect and resulted in a higher mortality rate. This higher mortality rate may be due to the greater abundance of baicalein and wogonin in HQD-2 than HQD-1. Furthermore, HQD-1 protected against DSS-induced chronic UC and significantly inhibited AOM/DSS-induced CRC in mice. HQD-1 also inhibited the production of inflammatory cytokines and increased antioxidant capacity both in chronic DSS and AOM/DSS treated mice. Overall, HQD-1 inhibits the development of acute/chronic colitis and prevents colitis-associated CRC, possibly by inhibiting inflammation and preventing oxidative stress induced cellular damage.

Mailuoning prevents high-glucose-mediated human umbilical vein endothelial cells apoptosis.

OBJECTIVEnTo investigated the role of Mailuoning in the prevention of high-glucose-mediated cell apoptosis in human umbilical vein endothelial cells (HUVEC) and the mechanisms involved.nnnMETHODSnMTT assay was used to investigate cell viability, western blot was used to investigate protein expression, and flow cytometric detection technology was used to detect cell apoptosis.nnnRESULTSnExposure of HUVEC to high glucose (50 mM) significantly suppressed cell viability and increased cell apoptosis compared with normal glucose (11 mM) (all P < 0.05). However, Mailuoning prevented high-glucose-induced HUVEC apoptosis in dose-dependent manner. Further studies indicated that Mailuoning suppressed high-glucose-induced p38 mitogen-activated protein kinase phosphorylation, but had no effect on extracellular signal-regulated kinase 1/2 and Akt phosphorylation.nnnCONCLUSIONnMailuoning can prevent high-glucose-induced HUVEC apoptosis by suppressing p38 activation.