AIM:To prepare a compound as the chemical reference substance of Guangjinqiancao Zonghuangtong Capsule.METHODS:To apply general column chromatography combined with preparative HPLC to isolate the target compound,to use analytic HPLC to determine the purity,stability and its content in the capsule,and to employ spectroscopic analysis (UV,IR,ESI-MS,1H-NMR,13C-NMR,DEPT,1H-13CCOSY,1H-1HCOSY,1DHOHAHA.1D.NOE,HMBC) to elucidate the structure of the isolated compound.RESULTS:The obtained compound was identified as isoschaftoside with the purity of over 99%, which was stable within 3 months at ambient temperature.As for isosehaftoside solution.it was stable within 8 h at ambient temperature.Its content in the capsule was above 3.0%.CONCLUSION:Isoschaftoside is a qualified reference substance for analytic assay ofGuangjinqiancao Zonghuangtong Capsule,and can be isolated from Desmodium styracifolium(Osb.)Merr.

Aim To detect the effects of cetuximab combined with adriamycin on the proliferation and ap-optosis of triple-negative breast cancer cells.Methods Cell viability was evaluated by MTT assay.The cell apoptosis was analyzed by flow cytometry with propidi-um iodide staining.JC-1 staining was used to deter-mine mitochondrial membrane potential.The expres-sions of glucose regulated protein78 (GRP-78),Bcl-2 and Caspase-3 were measured with Western blot.Re-sults MTT assay showed that cetuximab had inhibi-tion effect on the breast cancer cell MDA-MB-231 growth,and the effect was related to concentration of drug.The inhibition effect of adriamycin on MDA-MB-231 had remarkabe relationship with time and concen-tration.When combined with each other,they could re-markably increase inhibition effect.The viability of cells in combination group for 1 2 h,24 h,48 h,sig-nificantly lower than that in cetuximab or adriamycin group (P <0.05 or P <0.01 ).Apoptosis results showed that cell apoptosis was significantly increased when cetuximab combined with adriamycin,reached (43.93 ±3.59)% for 24 h,had remarkably statistical significance compared to cetuximab or adriamycin
group (P <0.01 ).JC-1 staining indicated that cetux-imab or adriamycin could reduce the mitochondrial membrane potential,but the reduction effect was more remarkable in the combination group.Western blot re-vealed that cetuximab could reduce the expression of GRP-78 and Bcl-2,and increased the expression of Caspase-3 and its activity.The expressions of Bcl-2, Caspase-3 had no significant change in adriamycin group,but GRP-78 was increased.In combination group,the expression of GRP-78 and Bcl-2 was signifi-cantly decreased,but Caspase-3 was increased nota-blely compared to adriamycin group.Conclusions The combination of cetuximab and adriamycin enhances the inhibition effect on the triple-negative breast cancer MDA-MB-231 cells,and increases cell apoptosis.The mechanism may be that cetuximab reduces the endo-plasmic reticulum stress level,then activates the mito-chondrial pathways by decreasing the expression of Bcl-2,reducing the mitochondrial membrane potential,and promoting cell apoptosis.

AIM: To develop an RP-HPLC method for preparing the reference substanc of vicenin-2 in Desmodium styracifolium. METHODS: Ethanol-extract of desmodium styracifolium was isolated and purified by RP-HPLC combining solvent extraction with column chromatography and recrystalliztion.The purity and content of vicenin-2 were identified by HPLC. RESULTS: The flvonoids were completely separated under this chromatographic condition.The purity of the reference substance was 99.0% or above. CONCLUSION: The method is simple,accurate,better on repeatability,and effective to yield high-purity product.It can be used as reference substance for the research of herbal medicine.

Objective: To evaluate the efficacy of Tuina (Chinese therapeutic massage) at points on abdomen and back meridians in the treatment of infantile colic.Methods: A total of 120 infants with intestinal colic were randomly divided into a control group and an observation group, with 60 cases in each group. In the control group, the parents of the infants were given soothing and health education. In addition to the intervention used in the control group, the observation group was treated with Tuina at points on abdomen and back meridians once a day for 5 consecutive days as a course of treatment. The pain scale score and clinical symptoms of the two groups were recorded before and after treatment. Results: After treatment, the total effective rate of the observation group was higher than that of the control group, and the difference was statistically significant (P<0.05). The pain scale score of the observation group was lower than that of the control group, and the difference was statistically significant (P<0.05). In the 24 h behavior diary indicators, the daily attack duration, the daily attack times, and the weekly attack days in the observation group were lower than those in the control group, and the differences were statistically significant (P<0.05). Conclusion: Tuina at points on abdomen and back meridians is effective and safe in the treatment of infantile colic.

OBJECTIVETo investigate the effect of low-molecular-weight heparin (LMWH) combined with paclitaxel (PTX) on the invasiveness and migration of nasopharyngeal carcinoma cells and explore the molecular mechanisms.

METHODSMTT assay was used to detect the growth inhibition induced by LMWH and PTX in CNE1 and CNE2 cells. Wound healing assay and Transwell migration assay were employed to assess the effects of the drugs on the cell migration, and Transwell invasion assay was used to evaluate the cell invasiveness. The cellular expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) were analyzed by Western blotting. ELISA was used to determine the expression of heparanase (HPA) in the culture medium of the cells.

RESULTSMTT assay showed an obvious suppression of CNE1 and CNE2 cell proliferation in response to LMWH and PTX treatments. Treatment with 200 U·ml LMWH combined with 0.1 µmol·L PTX for 24 h resulted in the inhibition rates of migration of 66.70% and 70.53% in CNE1 and CNE2 cells, respectively significantly higher than the rates in cells with PTX treatment alone. The combined treatment with LMWH and PTX for 24 h also caused a significantly higher inhibition rate of cell invasion than LMWH and PTX alone. LMWH enhanced the down-regulation of MMP-9 and HPA induced by PTX.

CONCLUSIONLMWH can enhance the inhibitory effect of PTX on the migration and invasion of nasopharyngeal carcinoma cells, the mechanism of which may involve the down-regulation of MMP-9 and HPA expressions.

Carcinoma ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Glucuronidase ; metabolism ; Heparin Lyase ; metabolism ; Heparin, Low-Molecular-Weight ; administration & dosage ; pharmacology ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Nasopharyngeal Neoplasms ; pathology ; Paclitaxel ; administration & dosage ; pharmacology ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism

OBJECTIVETo investigate the effect of chloroquine in reversing multidrug resistance (MDR) of HNE1/DDP cell line and explore the mechanism.

METHODSMTT assay was used to detect the cell viability of HNE1 and HNE1/DDP after exposure to different concentrations of DDP (2, 4, 8, 16 and 32 µmol/L) and different concentrations of chloroquine (5, 10, 20, 40, and 80 µmol/L). q-PCR was used to assess the expression of MDR1 mRNA and Western blotting was employed to detect P-glycoprotein (P-gp) expression in HNE1 and HNE1/DDP cells exposed to 5 and 10 µmol/L chloroquine. The cell apoptosis rate of HNE1 and HNE1/DDP cells exposed to 10 and 20 µmol

RESULTSChloroquine exposure caused dose-dependent suppression of the proliferation in both HNE1 and HNE1

CONCLUSIONChloroquine can reverse multidrug resistance in HNE1
ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Line, Tumor ; Chloroquine ; pharmacology ; Down-Regulation ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Humans

OBJECTIVETo explore the effect of the Hsp90 inhibitor anacardic acid on cell proliferation, invasion and migration of breast cancer MDA-MB-231 cells.

METHODSThe inhibitory effect of anacardic acid on Hsp90 was assessed with in vitro ATPase inhibition assay and ATP-sepharose binding assay. MTT assay was used to detect the growth inhibition induced by anacardic acid in MDA-MB-231 cells. Transwell assays were used to evaluate MDA-MB-231 cell invasion and migration. Western blotting was performed to assess the effect of anacardic acid in triggering the degradation of MMP-9, TIMP-1, Hsp90, and Hsp70.

RESULTSAnacardic acid exhibited a modest activity of ATPase inhibition with an IC50 value of 82.5 µmol/L. Anacardic acid significantly suppressed the proliferation of MDA-MB-231 cells in a dose-dependent manner (IC50 value of 29.3 µmol/L). Treatment with 12.5, 25, and 50 µmol/L anacardic acid for 36 h caused inhibition of cell invasion by 23.6%, 56.6%, and 67.0% in MDA-MB-231 cells, respectively (P<0.05), and anacardic acid treatment for 24 h inhibited the cell migration by 30.0%, 45.5%, and 77.5%, respectively (P<0.05). Anacardic acid dose-dependently induced MMP-9 degradation, but did not obviously affect Hsp90 or Hsp70 expressions.

CONCLUSIONAnacardic acid can significantly inhibit the proliferation, invasion, and migration of MDA-MB-231 cells, the mechanism of which may involve the inhibition of Hsp90 ATPse activity and down-regulation of MMP-9 expression.

Anacardic Acids ; pharmacology ; Breast Neoplasms ; pathology ; Cell Line, Tumor ; drug effects ; Cell Movement ; Cell Proliferation ; Down-Regulation ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; metabolism ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism

Objective To investigate the effects of metformin on the proliferation and apoptosis of human oral cancer cell line KB in vitro. Methods Human oral cancer cell line KB was exposed to different doses of metformin (0, 1.25, 2.5, 5, 10, and 20 mmol/L), and the changes in cell viability were detected using MTT assay. Colony formation of the cells was observed following an 8-day metformin exposure. The changes in mitochondrial membrane potential were measured by JC-1 assay, and PI staining was used to observe the cell apoptosis. Western blotting was employed to detect the changes in the protein expressions of GRP78 and activated caspase-3. Results Metformin exposure caused time-and dose-dependent suppression of KB cell proliferation, and exposure to 5 mmol/L metformin for 24, 48 and 72 h resulted in cell survival rates of 68.0%, 36.9%, and 14.5%, respectively. Metformin significantly inhibited KB cell colony formation. Exposure of the cells to increased concentrations of metformin gradually increased the apoptotic rate and decreased mitochondrial membrane potential. Metformin caused an initial up-regulation followed by a down-regulation of GRP78 expression in KB cells and increased the expression of activated caspase-3. Conclusion Metformin can inhibit the proliferation and induce apoptosis of KB cells, the mechanism of which may involve the activation of the mitochondrial apoptotic pathway and endoplasmic reticulum stress.

Objective To explore the effect of the Hsp90 inhibitor anacardic acid on cell proliferation, invasion and migration of breast cancer MDA-MB-231 cells. Methods The inhibitory effect of anacardic acid on Hsp90 was assessed with in vitro ATPase inhibition assay and ATP-sepharose binding assay. MTT assay was used to detect the growth inhibition induced by anacardic acid in MDA-MB-231 cells. Transwell assays were used to evaluate MDA-MB-231 cell invasion and migration. Western blotting was performed to assess the effect of anacardic acid in triggering the degradation of MMP-9, TIMP-1, Hsp90, and Hsp70. Results Anacardic acid exhibited a modest activity of ATPase inhibition with an IC50 value of 82.5 μmol/L. Anacardic acid significantly suppressed the proliferation of MDA-MB-231 cells in a dose-dependent manner (IC50 value of 29.3 μmol/L). Treatment with 12.5, 25, and 50μmol/L anacardic acid for 36 h caused inhibition of cell invasion by 23.6%, 56.6%, and 67.0%in MDA-MB-231 cells, respectively (P<0.05), and anacardic acid treatment for 24 h inhibited the cell migration by 30.0%, 45.5%, and 77.5%, respectively (P<0.05). Anacardic acid dose-dependently induced MMP-9 degradation, but did not obviously affect Hsp90 or Hsp70 expressions. Conclusion Anacardic acid can significantly inhibit the proliferation, invasion, and migration of MDA-MB-231 cells, the mechanism of which may involve the inhibition of Hsp90 ATPse activity and down-regulation of MMP-9 expression.

Objective To investigate the effect of chloroquine in reversing multidrug resistance (MDR) of HNE1/DDP cell line and explore the mechanism. Methods MTT assay was used to detect the cell viability of HNE1 and HNE1/DDP after exposure to different concentrations of DDP (2, 4, 8, 16 and 32μmol/L) and different concentrations of chloroquine (5, 10, 20, 40, and 80μmol/L). q-PCR was used to assess the expression of MDR1 mRNA and Western blotting was employed to detect P-glycoprotein (P-gp) expression in HNE1 and HNE1/DDP cells exposed to 5 and 10μmol/L chloroquine. The cell apoptosis rate of HNE1 and HNE1/DDP cells exposed to 10 and 20μmol/L chloroquine was determined by PI assay. Results Chloroquine exposure caused dose-dependent suppression of the proliferation in both HNE1 and HNE1/DDP cells, and significantly reversed multidrug resistance in HNE1/DDP cells. The expressions of MDR1 mRNA and P-gp protein were significantly lowered in the cells treated with chloroquine. Conclusion Chloroquine can reverse multidrug resistance in HNE1/DDP cells possibly through down-regulation of MDR1 and inhibition of P-gp protein.