BACKGROUND: Lung cancer is one of the highest morbidity and mortality in the world and it is very important to find an effective anti-tumor method. Microwave hyperthermia, a new treatment technology, has been getting more and more attention. This study was designed to investigate the effects of microwave hyperthermia combined with gemcitabine on the proliferation and apoptosis of human lung squamous cell carcinoma (NCI-H1703 and NCI-H2170) in vitro. METHODS: The proliferation of cells treated with microwave hyperthermia, the effect of gemcitabine on cell proliferation and the proliferation of cells treated with different methods of microwave hyperthermia and gemcitabine were detected by CCK-8 assay. Colony formation assay was used to measure the colony formation of human lung squamous cell carcinoma cells. Flow cytometry assay was used to detect the total apoptosis rates of the treated cells. Caspase-3, Caspase-8 activity assay was used to detect the activity of Caspase-3, Caspase-8 enzyme in each group of cells. CCK-8 assay was used to detect the effect of control group, AC-DEVD (Caspase-3 inhibitor) group, thermalization combined group, and thermal AC-DEVD combined group on cell proliferation. The levels of p53, Caspase-3, Cleaved-Caspase-3, PARP, Bax and BCL-2 protein expression were detected using Western blot assay. RESULTS: Our results demonstrated that microwave hyperthermia inhibited the proliferation of lung squamous cell carcinoma. The IC₅₀ values of gemcitabine for the two cells were 8.89 μmol/L and 44.18 μmol/L, respectively. The first chemotherapy after microwave hyperthermia has synergistic effect on the two lung squamous cell carcinoma cells and can significantly inhibit the cell clone formation (P<0.001), promote cell apoptosis (P<0.001) and increase Caspase-3 enzyme activity (P<0.001). However, it has no effect on Caspase-8 enzyme activity (P>0.05). Furthermore, Western blot analysis showed that microwave hyperthermia combined with gemcitabine could up-regulate the p53, Caspase-3, Cleaved-Caspase-3, Cleaved-PARP and Bax protein expression. CONCLUSIONS Microwave hyperthermia combined with gemcitabine remarkably inhibit the proliferation and induce apoptosis of human lung squamous cell carcinoma in vitro. This effect may be associated with the activation of p53, cleavage of PARP protein, and induced the Caspase-3 dependent apoptosis.
Apoptosis ; drug effects ; radiation effects ; Carcinoma, Squamous Cell ; pathology ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; radiation effects ; Combined Modality Therapy ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Humans ; Hyperthermia, Induced ; Lung Neoplasms ; pathology ; Microwaves

PURPOSE: This study was aimed to investigate the effect of pseudolaric acid B (PAB) on proliferation, invasion and epithelial-to-mesenchymal transition (EMT) in pancreatic cancer cells and to explore the possible mechanism. MATERIALS AND METHODS: The pancreatic cancer cell line SW1990 was cultured and treated with PAB dose- and time-dependent manners. Cell proliferation and invasion ability were measured by MTT assay and Matrigel/Transwell test, respectively. Semi-quantitative real-time polymerase chain reaction and Western blotting were conducted to detect the expression of EMT markers and the key molecules. Finally, nude mice subcutaneous transplantation tumor model was used to confirm the therapy efficacy of PAB. RESULTS: PAB could inhibit SW1990 cell proliferation and invasion in time- and dose-dependent manners. Vimentin, fibronectin, N-cadherin, Snail, Slug, YAP, TEAD1, and Survivin were down-regulated (p < 0.01), while E-cadherin, caspase-9, MST1, and pYAP were up-regulated (p < 0.05). Combined PAB and gemcitabine treatment markedly restricted the tumor growth compared with gencitabin or PAB alone groups. CONCLUSION: PAB could inhibit the proliferation and invasion ability of pancreatic cancer cells through activating Hippo-YAP pathway and inhibiting the process of EMT.
Animals ; Antineoplastic Agents/pharmacology ; Antineoplastic Agents/therapeutic use ; Biomarkers, Tumor/metabolism ; Cadherins ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation/drug effects ; Cytokines ; Deoxycytidine/analogs & derivatives ; Deoxycytidine/pharmacology ; Deoxycytidine/therapeutic use ; Diterpenes/pharmacology ; Diterpenes/therapeutic use ; Epithelial-Mesenchymal Transition/drug effects ; Female ; Humans ; Mice, Nude ; Neoplasm Invasiveness ; Pancreatic Neoplasms/diet therapy ; Pancreatic Neoplasms/pathology ; Real-Time Polymerase Chain Reaction ; Signal Transduction/drug effects ; Vimentin/metabolism

This study was aimed to investigate the effect of Honokiol (HNK) combined with Gemcitabine (GEM) on the proliferation and apoptosis of human Burkitt lymphoma Raji cells. Cell proliferation was detected by CCK-8 method to study the role of Honokiol and Gemcitabine in Raji cells. The cell apoptosis and cell cycle status were analyzed by flow cytometry. The level of apoptosis-related protein BCL-2 was measured with Western blot. The results showed that compared with cells treated with mentioned above drugs alone, the proliferative potential of cells in combination group was significantly inhibited (P < 0.01) and the inhibition rate was related to the concentration and action time of HNK; and apoptosis rate markedly increased (P < 0.01), while most Raji cells were arrested at G0/G1 phase and decreased in S phase after treatment with combination of two drugs; the expression of BCL-2 protein decreased (P < 0.01). It is concluded that Honokiol combined Gemcitabine can synergistically inhibit the proliferation, induce cell apoptosis, and down-regulate the expression of BCL-2 in Raji cells. The possible mechanism of synergistic effect may be related with arrest of cell cycle at G0/G1 phase and downregulation of the expression of BCL-2.
Apoptosis ; drug effects ; Biphenyl Compounds ; pharmacology ; Burkitt Lymphoma ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Drug Synergism ; Humans ; Lignans ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism

OBJECTIVETo investigate the effect of down-regulation of Notch1 by Notch1 small interfering RNA (siRNA) on chemosensitivity to gemcitabine in pancreatic cancer cells and its mechanism.

METHODSNotch1 siRNA was transfected to pancreatic cancer cell lines AsPC-1, BxPC-3, MIAPaCa-2 and Panc-1. The transfected pancreatic cancer cells were treated with 10 μmol/L gemcitabine in vitro. The relative quantity of Notch1 mRNA of pancreatic cancer cells was detected by real-time PCR. The inhibition rates of gemcitabine-treated cells were evaluated by CCK-8 method. The expression of Bax protein was examined by Western blot, and the caspase 3 activity was detected by CaspACETM assay system kit.

RESULTSThe relative quantity of Notch1 mRNA was the highest in BxPC-3 cell line and the lowest in Panc-1 cells. The inhibition rates of gemcitabine treated-cells were significantly higher in Notch1 siRNA transfection groups than in corresponding siRNA control groups (AsPC-1: 67.5±6.7 vs 47.5±6.8; BxPC-3: 90.5±4.4 vs 70.2±4.2; MIAPaCa-2: 80.9±5.7 vs 58.1±6.0; Ps<0.05), with the overexpression of protein Bax. The activity of caspase 3 was also significantly increased in Notch1 siRNA transfection groups compared with corresponding siRNA control groups (AsPC-1: 28.90±2.70 vs 12.82±3.44; BxPC-3: 59.87±6.77 vs 27.27±11.88; MIAPaCa-2: 29.34±4.06 vs 14.59±4.25; P<0.05).

CONCLUSIONInhibition of Notch signaling pathway by Notch1 siRNA can enhance chemosensitivity to gemcitabine in pancreatic cancer cells through activating apoptosis activity．

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Down-Regulation ; Humans ; Pancreatic Neoplasms ; pathology ; RNA, Small Interfering ; genetics ; Receptor, Notch1 ; genetics ; Signal Transduction ; bcl-2-Associated X Protein ; metabolism

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

This study was aimed to investigate the effect of methylation transferase inhibitor 5-aza-2'-deoxycytidine (5-aza-2dC) of different concentrations on the apoptosis of human acute myeloid leukemia (AML) cell line HL-60 and the expression of DAPK gene in HL-60 cells, as well as to explore the possible anti-AML mechanism of 5-aza-2dC. HL-60 cells were treated by 5-aza-2dC of different concentrations. The effect of 5-aza-2dC on the HL-60 cell morphology was observed by Wright's staining. The effect of 5-aza-2dC on HL-60 cell apoptosis and DAPK mRNA expression was detected by flow cytometry and reverse transcription-polymerize chain reaction (RT-PCR) respectively. The results showed that the 5-aza-2dC induced the apoptosis of HL-60 cells in a concentration-dependent manner; the 5-aza-2dC increased the expression levels of DAPK mRNA in HL-60 cells in a concentration-dependent manner. It is concluded that the apoptosis rate of HL-60 cells and DAPK mRNA expression level displayed a rising trend with 5-aza-2dC concentration increasing. Therefore, DAPK gene may participate in HL-60 cell apoptosis induced by 5-aza-2dC.
Apoptosis ; drug effects ; Death-Associated Protein Kinases ; genetics ; Deoxycytidine ; pharmacology ; Gene Expression ; drug effects ; HL-60 Cells ; Humans

OBJECTIVETo investigate the therapeutic efficacy of double-mutated oncolytic adenovirus AxdAdB-3 in combination with gemcitabine for treating bladder cancer in an orthotopic nude mouse model.

METHODSThe susceptibility to the adenovirus was evaluated in bladder cancer cell lines YTS-1, T24, 5637 and KK47, and normal cell lines HCV29 and WI38. The cells were infected with AxCAlacZ and stained with 5-bromo-4-chloro-3-indolyl-β-galactoside (X-Gal). Immunostaining against adenoviral hexon protein was performed to determine the selective replication of AxdAdB-3 in the cancer cells. Flow cytometry was used to determine the YTS-1 cells in S phase of cell cycle after adenovirus infection. Cell viability after AxdAdB-3 and/or gemcitabine was measured by CCK-8 assay. Orthotopic bladder cancer model was established in nude mice, and the inhibitory efficacy of intravesical instillation therapy with AxdAdB-3 or/and gemcitabine was assessed.

RESULTSGene transduction efficiency was different among the cell lines, and correlated with expression of CAR. 5637 and KK47 cells with high expression of CAR were more susceptible to the adenovirus, whereas YTS-1 and T24 cells with little CAR expression were resistant to adenoviral infection. Immunostaining showed that the expression levels of hexon protein varied among the cell lines. Normal cells infected with AxdAdB-3 expressed little hexon protein. The proportion of S-phase cells was (39 ± 3) % and (49 ± 5) % in the AxCAlacZ- and AxdAdB-3-infected bladder cancer cells, respectively. AxdAdB-3 effectively induced S-phase entry of cell cycle (P < 0.05). AxdAdB-3 combined with gemcitabine significantly inhibited the growth of bladder cancer cell lines. In vivo, the mean weight of the bladder tumors in mice treated with intravesical instillation of AxCAlacZ, gemcitabine, AxdAdB-3, and AxdAdB-3 + gemcitabine were 400.6, 126.4, 82. 0, 40.4 mg, respectively. Either AxdAdB-3 (P < 0.0001) and gemcitabine (P < 0.0001) suppressed the tumor growth in nude mice, and the combination therapy reduced tumors more effectively than either AxdAdB-3 (P < 0.0001) or gemcitabine (P < 0.0001) alone.

CONCLUSIONSIntravesical instillation therapy with AxdAdB-3 in combination with gemcitabine can effectively inhibit the orthotopic bladder cancer in nude mouse, and further relevant clinical studies are guaranteed.

Adenoviridae ; genetics ; Administration, Intravesical ; Animals ; Antimetabolites, Antineoplastic ; administration & dosage ; pharmacology ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; administration & dosage ; pharmacology ; therapeutic use ; Deoxycytidine ; administration & dosage ; analogs & derivatives ; pharmacology ; therapeutic use ; Galactosides ; Indoles ; Mice ; Mice, Nude ; Models, Animal ; Urinary Bladder Neoplasms ; drug therapy

BACKGROUND/AIMS: Heat shock protein (HSP) 70 is constitutively overexpressed in pancreatic cancer cells (PCCs) and appears to confer protection against chemotherapeutics. We investigated whether modulating HSP 70 increases chemoresponsiveness to gemcitabine in PCCs. METHODS: Varying concentrations of quercetin and gemcitabine, either alone or in combination, were added to PCCs (Panc-1 and MiaPaCa-2). MTT assay was performed to analyze cell viability. HSP 70 expression was assessed by Western blot analysis. Apoptosis was determined by measuring caspase-3 activity. Western blot for the LC3-II protein detected the presence of autophagy. RESULTS: HSP 70 levels were not affected by the incubation of Panc-1 and MiaPaCa-2 cells with gemcitabine, whereas with quercetin, the levels were reduced in both cell lines. The viability of both Panc-1 and MiaPaCa-2 cells significantly decreased with gemcitabine treatment but not with quercetin. A combination of gemcitabine and quercetin decreased the viability of both cell lines in a dose-dependent manner, which was more pronounced than gemcitabine treatment alone. Treatment with either gemcitabine or quercetin augmented caspase-3 activity in both cell lines, and a combination of these compounds further potentiated caspase-3 activity. LC3-II protein expression was negligible with gemcitabine treatment but marked with quercetin. The addition of gemcitabine to quercetin did not potentiate LC3-II protein expression. CONCLUSIONS: Modulation of HSP 70 expression with quercetin enhanced the chemoresponsiveness of PCCs to gemcitabine. The mechanism of cell death was both apoptosis and autophagy.
Antineoplastic Combined Chemotherapy Protocols/*pharmacology ; Apoptosis/drug effects ; Autophagy/drug effects ; Caspase 3/metabolism ; Cell Line, Tumor ; Cell Survival/*drug effects ; Deoxycytidine/analogs & derivatives/pharmacology ; Drug Resistance, Neoplasm/*drug effects ; HSP70 Heat-Shock Proteins/*metabolism ; Humans ; Microtubule-Associated Proteins/metabolism ; Pancreatic Neoplasms/*drug therapy ; Quercetin/pharmacology

OBJECTIVETo study the effect of emodin combined gemcitabine on the growth of pancreatic cancer in vivo and in vitro as well as its mechanisms.

METHODSAfter human pancreatic cancer cell line SW1990 was treated with emodin (40 micromol/L), gemcitabine (20 micromol/L), and emodin combined gemcitabine, the cell proliferation was detected by cell counting kit-8 (CCK-8) assay. The apoptosis of pancreatic cancer cells was detected using the flow cytometry (FCM). The protein expressions of Bax and Bcl-2 were detected using Western blot. SW1990 cells were injected subcutaneously into nude mice to establish pancreatic xenograft tumors. The mice were then treated by emodin, gemcitabine, and emodin combined gemcitabine, respectively. The changes of tumor volume were monitored. The positive expressions of Ki-67, Bax, and Bcl-2 in the xenograft tumors were detected using immunohistochemical method.

RESULTSEmodin combined with gemcitabine induced a higher percentage of growth inhibition and apoptosis in pancreatic cancer cell line SW1990 than that of gemcitabine or emodin alone (P < 0.05). The protein expression of Bax was up-regulated and that of Bcl-2 down-regulated in the emodin group and the emodin combined gemcitabine group when compared with the control group (P < 0.05). Emodin combined with gemcitabine could significantly inhibit the growth of pancreatic xenograft tumors, increase the positive expression of Bax in tumor tissues, obviously decrease the positive expressions of Ki-67 and Bcl-2 (P < 0.05). The optimal effects were obtained in the emodin combined gemcitabine group (P < 0.05).

CONCLUSIONEmodin could potentiate the inhibition of pancreatic cancer growth induced by gemcitabine both in vitro and in vivo, which might be achieved by up-regulating the expression of Bax and down-regulating the expression of Bcl-2.

Animals ; Cell Line, Tumor ; Cell Proliferation ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Emodin ; pharmacology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Ki-67 Antigen ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Pancreatic Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; metabolism

Sapacitabine is an orally bioavailable prodrug of the nucleoside analog 2'-C-cyano-2'-deoxy-1-β-D-arabino-pentofuranosylcytosine (CNDAC). Both the prodrug and active metabolite are in clinical trials for hematologic malignancies and/or solid tumors. CNDAC has a unique mechanism of action: after incorporation into DNA, it induces single-strand breaks (SSBs) that are converted into double-strand breaks (DSBs) when cells go through a second S phase. In our previous studies, we demonstrated that CNDAC-induced SSBs can be repaired by the transcription-coupled nucleotide excision repair pathway, whereas lethal DSBs are mainly repaired through homologous recombination. In the current work, we used clonogenic assays to compare the DNA damage repair mechanism of CNDAC with two other deoxycytidine analogs: cytarabine, which is used in hematologic malignacies, and gemcitabine, which shows activity in solid tumors. Deficiency in two Rad51 paralogs, Rad51D and XRCC3, greatly sensitized cells to CNDAC, but not to cytarabine or gemcitabine, indicating that homologous recombination is not a major mechanism for repairing damage caused by the latter two analogs. This study further suggests clinical activity and application of sapacitabine that is distinct from that of cytarabine or gemcitabine.
Animals ; Antimetabolites, Antineoplastic ; pharmacology ; Arabinonucleosides ; pharmacology ; CHO Cells ; Cricetinae ; Cricetulus ; Cytarabine ; analogs & derivatives ; pharmacology ; Cytosine ; analogs & derivatives ; pharmacology ; DNA Breaks, Double-Stranded ; drug effects ; DNA Repair ; drug effects ; DNA-Binding Proteins ; deficiency ; Deoxycytidine ; analogs & derivatives ; pharmacology ; Homologous Recombination ; genetics ; Inhibitory Concentration 50 ; Prodrugs