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To investigate the expressions of the pannexin (Panx) proteins in I-10 Leydig tumor cells and TM3 Leydig cells and their regulatory effect on the Panx channel function in mice.The expressions of the Panx-1 and Panx-2 proteins in the mouse Leydig tumor cells were determined by Western blot. The I-10 Leydig tumor cells were treated with carbenoxolone (CBX) at 100 μmol/L or probenecid (PBN) at 200 μmol/L, the fluorescence resonance energy transfer (FRET) detected by time-lapse fluorescence imaging, and the extracellular adenosine 5'-triphosphate (eATP) level measured with the commercial detection kit. Molecular biological methods were used to interfere with shRNA and overexpress mPanx-1 the Panx-1 gene and regulate the expression and function of the Panx-1 protein.The expressions of Panx-1 (［289.5 ± 55.8］%) and Panx-2 (［264.5 ± 24.6］%) were significantly increased in the I-10 Leydig tumor cells as compared with those in the normal TM3 Leydig cells (both P0.05). FRET was remarkably reduced after treated with CBX (［87.5 ± 17.7］%) and PBN (［89.3 ± 14.3］%) in comparison with that in the control group (both P0.01). At 8, 16 and 24 hours, the eATP level was decreased by (57.3 ± 7.2)%, (56.4 ± 9.6)% and (63.4 ± 6.4)% in the CBX group (P0.01) and (61.7 ± 2.5)%, (35.8 ± 1.6)% and (13.5 ± 8.3)% in the PBN group (P0.01). Molecular biological treatment down-regulated the expression of Panx-1 by (38.3 ± 5.2)% and (31.8 ± 5.1)% in the shRNA1 and shRNA2 groups, respectively (both P0.01), but up-regulated that of Panx-1 by (128.4 ± 7.5)% in the mPanx-1 group (P0.01) as compared with the negative control. FRET was reduced by (72.4 ± 39.4)% in the shRNA group (P0.01) and the eATP level by (14.7 ± 0.1)%, (13.7 ± 0.3)% and (13.1 ± 0.3)% at 8, 16 and 24 hours, respectively (P0.01) while FRET elevated by (122.5 ± 17.1)% in the mPanx-1 group (P0.01) and the eATP level by (886.1 ± 82.1)%, (885.8 ± 83.3)% and (841.5 ± 21.8)% at 8, 16 and 24 hours, respectively (P0.01).The expressions of Panx-1 and Panx-2 are increased in I-10 mouse Leydig tumor cells, and inhibiting the Panx channel with CBX, PBN and shRNA reduces FRET and the eATP level in the I-10 cells.

To investigate the role of SRC kinase inhibitor PP2 in drug resistance to adriamycin (ADM) in breast cancer cells and invasion, metastasis of cells.MTT assay was used to detect the inhibitory effect of ADM on MCF-7 and MCF-7/ADM cells. The 50% inhibitory concentration (ICADM inhibited the proliferation of MCF-7 more than MCF-7/ADM cells (The resistance of MCF-7 to ADM is accompanied by increased expression of SRC. SRC inhibitor PP2 can reduce the cell resistance, ability of invasion and metastasis.

Previous studies have shown that irinotecan (CPT‑11) impairs chemotherapy‑induced apoptosis by activating nuclear factor‑κB (NF‑κB) and a number of strategies have been employed to augment chemosensitivity through the suppression of NF‑κB activation. Berberine, a botanical alkaloid, was reported to enhance chemosensitivity to 5‑fluorouracil and doxorubicin by suppressing NF‑κB activation. In the present study, the effect of berberine on CPT‑11‑induced apoptosis was investigated through the inhibition of NF‑κB. Inhibition of NF‑κB activation by p65 small interfering RNA was shown to potentiate apoptosis induced by CPT‑11. Berberine suppressed CPT‑11‑induced NF‑κB activation in a dose‑dependent manner and enhanced chemosensitivity to CPT‑11 by downregulating NF‑κB activation of antiapoptotic genes, c‑IAP1, c‑IAP2, survivin and Bcl‑xL. The current observations indicate that berberine inhibits NF‑κB activation and may be used to enhance CPT‑11‑induced apoptosis in colon cancer.

To investigate the role of pannexin 1 channels in cisplatin-induced apoptosis in I-10 cells and the mechanisms.MTT assay was used to assess the cytotoxicity of cisplatin (DDP) in I-10 cells. Annexin V/PI double staining and Hoechst 33258 fluorescence staining were employed to detect early- and late-stage apoptosis of the cells, respectively. Extracellular ATP level and intracellular IP3 level in the cells were detected using commercial detection kits.I-10 cells exposed to both CBX (a pannexin 1 channel inhibitor) and DDP showed a higher cell viability compared with the cells exposed to DDP alone (P0.01). CBX significantly decreased cisplatin-induced early-stage apoptosis (P0.001) and late-stage apoptosis (P0.01), and cause obvious reductions in extracellular ATP and intracellular IP3 levels during cisplatin-induced apoptosis (P0.05).Pannexin 1 channels participate in cisplatin-induced apoptosis in I-10 cells possibly through the ATP/IP3 pathway.

Panax Notoginseng Saponins (PNS) have been well known to have anti-tumor activity and enhance cytotoxicity of some cancer chemotherapy agents, but the mechanisms underlying these effects are still unknown. This study investigates the effect of PNS on cytotoxicity of cisplatin and the relationship between this effect and the modulation of gap junctions (GJ) function by PNS in a transfected cell line. The cytotoxicity of cisplatin (0.25-1 µg/mL) was increased in the presence of GJ. Inhibition of gap junction by either GJ blocker or interception of Connexin (Cx) expression decreased the cytotoxicity of cisplatin. Increasing GJ function enhanced cytotoxicity of cisplatin, only in the cells with functional GJ. PNS (50-200 µg/mL) significantly enhanced cisplatin cytotoxicity, but this effect required functional gap junctions between the cells. Exposure of the cells to PNS (50-200 µg/mL) for 4 h leads to a significant enhance in dye coupling of GJ in a dose-dependent manner. These results suggest that PNS increases the cytotoxicity of cisplatin through enhancement of GJ activity.

To observe the inhibitory effect of gefitineb on the proliferation and its inducing effect on the apoptosis of mouse I-10 Leydig testicular cancer cells in vitro.We treated I-10 Leydig testicular cancer cells of mice with gefitineb at 0, 1.25, 2.5, 5, 10, 20, and 40 µmol/L. Then we determined the inhibitory effect of gefitineb on the growth of the cells by MTT, detected their early and late apoptosis by Annexin V-FITC/propidium iodide double staining and Hoechst 33258 nuclear staining, respectively, and observed the expressions of apoptosis-related proteins Bcl-2, Bax and caspase 3/9 by Western blot.Compared with the blank control group, gefitineb significantly inhibited the proliferation of the I-10 cells at 10 and 20 µmol/L (P0.05). The survival rate of the cells was (32.4 ± 2.8)% (P0.01) and their early and late apoptosis rates were (26.7 ± 4.2)% and (59.33 ± 10.2)% in the 40 µmol/L group, significantly different from those in the control (P0.05 and P0.01). In comparison with the blank control group, gefitineb at 10, 20, and 40 µmol/L increased the expression of pro-apoptotic protein Bax by (41.9 ± 7.1), (60.1 ± 9.8), and (69.0 ± 11.3)% (all P0.05), decreased that of apoptosis-inhibitory protein Bcl-2 by (50.3 ± 8.9), (63.9 ± 6.9), and (88.7 ± 13.9)% (all P0.05), and elevated that of the cleft proteins caspase-3 by (69.0 ± 6.9)% (P0.05), (71.5 ± 8.1)% (P0.05), and (110.9 ± 14.2)% (P0.01) and caspase-9 by (51.8 ± 4.9), (54.7 ± 6.7), and (43.8 ± 11.8)% (all P0.05).Gefitineb can increase the cytotoxicity of I-10 Leydig testicular cancer cells of mice and induce their apoptosis via the mitochondria-mediated apoptosis signaling pathway.

To investigate the effect of baicalein on the gap junction intercellular communication (GJIC) in the TM4 Sertoli cells of the mouse testis and its related mechanism.We measured the cytotoxicity of different concentrations of baicalein on the TM4 Sertoli cells in the mouse testis by MTT, detected the fluorescence transfer of the TM4 Sertoli cells by parachute assay, and determined the expression of the protein connexin 43 ( Cx43) in the baicalein-treated cells by Western blot and immunofluorescence assay.Baicalein produced no obvious cytotoxicity on the TM4 Sertoli cells at the concentration below 60 µmol/L but significantly increased their GJIC at 0-20 µmol/L (P0.01). Western blot and immunofluorescence assay showed that 0-20 µmol/L baicalein remarkably elevated the expression of Cx43 in the TM4 cells (P0.01) and on the membrane of the TM4 cells.Baicalein at the concentration of 0-20 µmol/L can significantly enhance GJIC in mouse TM4 Sertoli cells by increasing the expression of the Cx43 protein.

To investigate the effects of inhibiting gap junctional intercellular communication on hypoxia/reoxygenation injury in astrocytes.Primary cultured cerebral cortical astrocytes of neonate rats were divided into normal control group, hypoxia reoxygenation injury group and 18-α-glycyrrhetinic acid and oleamide (gap junctional intercellular channel inhibitors) group. The gap junction intercellular communication was determined by Parachute assay. The viability of astrocyes was detected by MTT assay. The apoptosis of astrocytes were detected with annexin V/PI and Hoechst 33258 staining.Compared with the normal control group, the gap junctional function of astrocytes was increased significantly in ischemia/reperfusion group (P0.01), the surviving fraction of astrocytes decreased significantly (P0.01) and its cell apoptosis ratio increased significantly (P0.01). Compared with the ischemia/reperfusion group, the gap junctional function of astrocytes in18-α-glycyrrhetinic acid and oleamide group decreased significantly (P0.01), the viability of astrocytes increased significantly (P0.01), while cell apoptosis decreased significantly (P0.01).Inhibition of intercellular gap junction has protective effect against hypoxia/reoxygenation injury in astrocytes.

To investigate the effect of sodium valproate, a histone deacetylase inhibitor, on the cytotoxicity of doxorubicin in breast cancer cells.Western blotting was used to assess Cx43 protein expression in breast cancer Hs578T cells exposed to doxorubicin and sodium valproate. MTT assay was used to determine the cytotoxicity of doxorubicin; annexin V/PI double staining and Hochest 33258 fluorescence staining were employed to detect doxorubicin-induced early and late apoptosis, respectively.Western blotting showed that sodium valproate significantly increased Cx43 protein expression in Hs578T cells (P/0.01). The cells exposed to both sodium valproate and doxorubicin showed significantly lowered cell viability compared with the cells exposed to doxorubicin alone (P/0.01). Exposure to both sodium valproate and doxorubicin resulted in significantly increased early and late cell apoptosis rate compared with doxorubicin treatment alone (P/0.01).sodium valproate can significantly enhance the cytotoxicity of doxorubicin and increase doxorubicin-induced apoptosis in breast cancer cells in vitro possibly by enhancing the gap junction function.

To investigate the effects of total flavonoids of Litsea Coreana (TFLC) on the gap junction (GJ) intercellular communication in TM3 testicular Leydig cells and whether TFLC can reduce the cytotoxicity of oxaliplatin (OHP) in vitro.We detected the effect of TFLC on the dye spread of the in vitro cultured TM3 cells by parachute assay, observed changes in the expression of connexin 43 (Cx43) total protein in the TFLC-treated TM3 cells by Western blot, and determined the effects of TFLC on the expression of Cx43 on the membrane of the TM3 cells by immunofluorescence assay and on the cytotoxicity of OHP by MTT assay.TFLC obviously enhanced the GJ function with the increasing of the TFLC concentration in the TM3 cells. Western blot and immunofluorescence assay confirmed that TFLC significantly enhanced the expression of Cx43 total protein and Cx43 expression on the membrane of the TM3 cells. MTT assay showed that at a high cell density (confluent with GJ formation), 20 microg/ml TFLC enhanced the GJ function of the TM3 cells and reduced the cytotoxicity of OHP (P0.05), while at a low density (preconfluent with no GJ formation), TFLC exhibited no effect on the cytotoxicity of OHP (P0.05).TFLC increases the Cx43 expression and GJ function in normal TM3 Leydig cells, and the enhancement of GJ function reduces the cytotoxicity of OHP.

To investigate the effects of retinoic acid (RA) on the expression of Cx43 and its gap junction intercellular communication function in testicular cancer cells.Cultured testicular cancer cells I-10 were treated with different concentration of RA (2.5, 5.0,10.0 micromol/L). The expression of Cx43 in 1-10 cells was detected by Western blot, and the distribution and location of Cx43 on cellular membrane was studied with immunofluorescence assay. Parachute assay was used to detect the function of gap junction intercellular communication composed of Cx43 in 1-10 cells.RA (2.5, 5.0, 10.0 micromol/L) markedly increased the expression of Cx43 in I-10 cells, the enhancement ratios were 43.14% +/- 2.1%, 58.09% +/- 1.8%, 143.13% +/- 1.6%, respectively. The result of immunofluorescence assay showed that RA (2.5, 5.0, 10.0 micromol/L) obviously increased the level of Cx43 located on the cellular membrane of I-10 cells. The result of parachute assay demonstrated that RA (2.5,5.0,10.0 gmol/L) could enhance the intercellular dye coupling through gap junction, the enhancement ratios were 26.1% +/- 2.3%, 63.3% +/- 1.6%, 140.5% +/- 3.4%, respectively.RA could enhance the gap junction intercellular communication by increasing the expression of Cx43 in I-10 cells.

Panax Notoginseng Saponins (PNS) have been well known to have anti-tumor activity and enhance cytotoxicity of some cancer chemotherapy agents, but the mechanisms underlying these effects are still unknown. This study investigates the effect of PNS on cytotoxicity of cisplatin and the relationship between this effect and the modulation of gap junctions (GJ) function by PNS in a transfected cell line. The cytotoxicity of cisplatin (0.25-1 µg/mL) was increased in the presence of GJ. Inhibition of gap junction by either GJ blocker or interception of Connexin (Cx) expression decreased the cytotoxicity of cisplatin. Increasing GJ function enhanced cytotoxicity of cisplatin, only in the cells with functional GJ. PNS (50-200 µg/mL) significantly enhanced cisplatin cytotoxicity, but this effect required functional gap junctions between the cells. Exposure of the cells to PNS (50-200 µg/mL) for 4 h leads to a significant enhance in dye coupling of GJ in a dose-dependent manner. These results suggest that PNS increases the cytotoxicity of cisplatin through enhancement of GJ activity.

To observe the influence of Cx26/Cx32 gap junction channel on the antineoplastic effect of etoposide in Hela cervical cancer cells.Fluorescence trace was used to assay the gap junction intercellular communication mediated by Cx26/Cx32 in Hela cells and its functional modulation by the pharmacological agents (oleamide, retinoid acid). A standard colony-forming assay was applied to determine the cell growth-inhibiting effect of etoposide in Hela cells with functional modulation of the gap junction. Hoechst 33258 staining was used to assess the changes in etoposide-induced apoptosis of Hela cells with altered gap junction functions.Oleamide markedly decreased while retinoid acid obviously increased the gap junction function in Hela cells. Standard colony-forming assay showed that etoposide produced a lowered antiproliferative effect in Hela cells with reduced gap junction and an increased antiproliferative effect in cells with enhanced gap junction function. In cells with a reduced gap junction function, etoposide induced a lowered apoptosis rate, which increased obviously in cells with an enhanced gap junction function.The antineoplastic effect of etoposide is reduced in Hela cells with a decreased gap junction intercellular communication mediated by Cx26/Cx32 and is enhanced in cells with an increased gap junction intercellular communication.