OBJECTIVE: To investigate the effect of SPARC gene overexpression on the chemotherapeutic sensitivity of AML-MDS cell line SKM-1 to Ara-C and to further explore its mechanism. METHODS: Subjects were divided into 6 groups: SKM-1 cells (Control), Negative control (LV-NC), SPARC overexpression (LV-SPARC), SKM-1 cells+30 ng/ml Ara-C (30 ng/ml Ara-C), LV-NC+30 ng/ml Ara-C and LV-SPARC+30 ng/ml Ara-C. Cell activity was detected by CCK-8 assay, cell cycle distribution and apoptosis were detected by flow cytometry, mRNA expression levels of SPARC, CPBP and MLKL were detected by RT-qPCR, and the expression levels of related protein were detected by Western blot. RESULTS: After co-treatment with SPARC overexpression and Ara-C, the cell viability decreased and apoptosis increased significantly, with obvious up-regulation of Bax and down-regulation of BCL-2 (P<0.05). Compared with the control group, the cell cycle of LV-SPARC+30 ng/ml Ara-C group was significantly arrested in S phase with obvious down-regulation of CDK2 and up-regulation of p27^KIP1 (P<0.05). Compared with LV-SPARC group and 30 ng/ml Ara-C group, the mRNA and protein expression levels of CPBP and MLKL (p-MLKL) were significantly elevated in LV-SPARC+30 ng/ml Ara-C group (P<0.05). In addition, after co-treatment with SPARC overexpression and Ara-C, the protein expression level of p-AKT decreased and the protein expression level of p53 increased (P<0.05). CONCLUSION SPARC overexpression enhanced the sensitivity of SKM-1 cells to Ara-C and promoted cell cycle arrest and apoptosis, the mechanism of which may be related to the regulation of CPBP/MLKL pathway.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Cytarabine ; Humans ; Kruppel-Like Factor 6/metabolism* ; Osteonectin/pharmacology* ; Protein Kinases/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; RNA, Messenger ; Sincalide/pharmacology* ; Tumor Suppressor Protein p53 ; bcl-2-Associated X Protein/pharmacology*

OBJECTIVEThe aim of this study was to explore the effect of SPARC on the anti-cancer effect of gemcitabine and underlying mechanism in pancreatic cancer.

METHODSAfter treating with gemcitabine, the proliferation rate of MIA PaCa2, MIA PaCa2/V and MIA PaCa2/SPARC69 cells was detected by MTT assay. The cell cycle distribution and cell apoptosis in each group were examined by flow cytometry, and the capability of clone formation was tested by adhesion-dependent clone formation assay. The apoptosis-related proteins were analyzed by Western blot.

RESULTSThe growth of pancreatic cancer cells was inhibited by gemcitabine in a time-dependent and dose-dependent manner. Its IC50 at 24, 48, and 72-h was (40.1 ± 2.5) µmol/L, (15.0 ± 0.5) µmol/L and (6.6 ± 0.1) µmol/L, respectively. The overexpression of SPARC increased the inhibitory effect of gemcitabine on growth of pancreatic cancer MIA PaCa2/SPARC69 cells, presenting a dose- and time- dependent manner. Its IC50 at 24, 48, 72 h was (24.3 ± 1.5) µmol/L, (7.7 ± 0.3) µmol/L and (4.8 ± 0.2) µmol/L, respectively. The clone formation assay showed that before gemcitabine treatment, the clone numbers of MIA PaCa2, MIA PaCa2/V and MIA PaCa2/SPARC69 cells were (2350 ± 125), (2130 ± 120) and (1567 ± 11), respectively. After gemcitabine treatment, the clone numbers of MIA PaCa2, MIA PaCa2/V and MIA PaCa2/SPARC69 cells were ( 1674 ± 79) , (1587 ± 94) and (557 ± 61), respectively. The overexpression of SPARC enhanced the chemosensitivity of MIA PaCa2 cells to gemcitabine chemotherapy. After treating with 10 µmol/L gemcitabine for 48 h, the ratio of G0/G1 cells in MIA PaCa2, MIA PaCa2/V and MIA PaCa2/SPARC69 cells were (56.0 ± 5.5)%, (55.0 ± 4.5)% and (68.0 ± 7.0)%, respectively. The cells arrested at G0/G1 phase were significantly increased in the MIA PaCa2/SPARC69 cells. The apoptosis rates of MIA PaCa2, MIA PaCa2/V and MIA PaCa2/SPARC69 cells were (22.4 ± 2.5)%, (19.9 ± 2.0)% and (37.7 ± 3.9)%, respectively, indicating that overexpression of SPARC enhanced the gemcitabine-induced apoptosis in MIA PaCa2 cells. The Western blot analysis showed that, compared with MIA PaCa2 and MIA PaCa2/V cells, the expression of caspase-2, -8, -9 and cleaved PARP protein was significantly increased, while the expression of Bcl-2 was not changed significantly in the MIA PaCa2/SPARC69 cells.

CONCLUSIONSPARC can enhance the chemosensitivity of pancreatic cancer cells to gemcitabine via regulating the expression of apoptosis-related proteins.

Antimetabolites, Antineoplastic ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Caspase 2 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Cell Cycle ; drug effects ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cysteine Endopeptidases ; metabolism ; Deoxycytidine ; administration & dosage ; analogs & derivatives ; pharmacology ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm ; Humans ; Osteonectin ; metabolism ; Pancreatic Neoplasms ; metabolism ; pathology ; Poly(ADP-ribose) Polymerases ; metabolism ; Time Factors

OBJECTIVETo investigate the effect of one kind of regulating-qi and Nourishing-yin Chinese herbs Zuoguiyin on the expression of rats ovarian vascular endothelial growth factor (VEGF) and secreted protein acidic rich in cysteine (SPARC) during the period of peri-menopause.

METHODThe animal models of perimenopause rats were established by natural aging. Perimenopause rats were treated by intragastric administration (ig) with low (13.78 g x kg(-1)), middle (20.67 g x kg(-1)) and high (31 g x kg(-1)) dose of Zuoguiyin for 8 weeks. Expression of VEGF and SPARC mRNA were detected by RT-PCR. Immunohistochemistry was used to evaluate expression levels of VEGF protein.

RESULTCompared with that in the control group, ovarian expression levels of VEGF mRNA and its protein in rats during peri-menopause increased significantly (P < 0.01). Middle and high dose of Zuoguiyin could both down-regulate the expression of VEGF in ovaries of praesenilis rats, and the difference has statistical significance (P < 0.01). Expression levels of SPARC mRNA in rat ovaries during peri-menopausal period decreased obviously compared to that in the control group (P < 0.01). Middle and high dose of Zuoguiyin could greatly promote ovarian SPARC mRNA expression of praesenilis rats (P < 0.01).

CONCLUSIONThe abnormal changes of VEGF and SPARC may play an important role in the aging process of ovary. Zuoguiyin, one kind of regulating-qi and Nourishing-yin Chinese herbs, can improve ovarian vascular formation through down-regulating the expression of VEGF meanwhile up-regulating the expression of SPARC. And it may delay aging by this way.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gene Expression ; drug effects ; Humans ; Models, Animal ; Osteonectin ; genetics ; metabolism ; Ovary ; drug effects ; metabolism ; Perimenopause ; drug effects ; metabolism ; Rats ; Rats, Wistar ; Vascular Endothelial Growth Factor A ; genetics ; metabolism