RASSF1A suppresses proliferation of cervical cancer cells.

BACKGROUND: This study aimed to explore the effects of ras association domain family 1 A (RASSF1A) on proliferation and apoptosis of human cervical cancer cell line Hela cells. MATERIALS AND METHODS: RASSF1A was cloned into the pcDNA3.1(+) vector to generate pcDNA3.1(+)-RASSF1A plasmid for transfection into Hela cells. Changes in the proliferation and apoptosis of cultured Hela cells were examined by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium chloride assay and flow cytometry. A protein array was used to analyze the expression of apoptotic factors. RESULTS: Plasmid pcDNA3.1(+)-RASSF1A was generated and transfected into Hela cells to stably express RASSF1A in Hela cells. RASSF1A transfection was effective in inhibiting the proliferation of Hela cells up to 52.4%, as compared to cells transfected with an empty plasmid. RASSF1A expression also successfully induced apoptosis in human cervical cells with an apoptosis rate of 20.5%. More importantly, protein array results showed that RASSF1 A transfection induced overexpression of p21 and caspase 8, while decreasing the expression of survivin in Hela cells. CONCLUSIONS: RASSF1A expression was effective in suppressing the proliferation and increasing apoptosis of Hela cells, and may be a potential therapy for cervical cancer in clinic.

Multiple mechanisms underlying the long duration of action of thienorphine, a novel partial opioid agonist for the treatment of addiction.

AIMS: It is considered that a long-acting therapy would be advantageous in the treatment of addiction. In a search for novel buprenorphine analogues, thienorphine was demonstrated to be an extremely long-acting orally active partial opioid agonist. This study explored the mechanisms underlying the long-lasting effects of thienorphine. METHODS: The binding kinetics of [(3) H]thienorphine were measured in membrane preparations expressing cloned rat opioid receptors. Flow cytometric analysis was used to determine the effect of thienorphine on the surface opioid receptor number. The long-lasting effects of thienorphine were also confirmed at the tissue level and in vivo. RESULTS: At 37°C, [(3) H]thienorphine showed rapid association with µ- and κ-opioid receptors, while its dissociation was sluggish and biphasic (K-1 = 0.21 min(-1) , K-2 = 0.0078 min(-1) for the µ-receptor; K-1 = 0.17 min(-1) , K-2 = 0.0042 min(-1) for the κ-receptor). Treatment with thienorphine for 24, 48, and 72 h downregulated surface µ-receptor in a dose- and time-dependent manner. The inhibitory effect of thienorphine on guinea pig ileum persisted for more than 120 min after prolonged washing. In vivo, thienorphine exhibited significant antagonism of morphine-induced antinociception for more than 7 days. CONCLUSIONS: These results indicate that multiple factors, including persistent receptor occupation and enhanced receptor downregulation, may contribute to the long-lasting effects of thienorphine that would be beneficial for its application in addiction treatment.

Effect of thienorphine on intestinal transit and isolated guinea-pig ileum contraction.

AIM: To evaluate the effect of thienorphine on small intestinal transit in vivo and on guinea-pig ileum (GPI) contraction in vitro. METHODS: The effects of thienorphine on intestinal transit were examined in mice and in isolated GPI. Buprenorphine and morphine served as controls. The distance traveled by the head of the charchol and the total length of the intestine were measured in vivo. Gastrointestinal transit was expressed as a percentage of the distance traveled by the head of the marker relative to the total length of the small intestine. The isolated GPI preparations were connected to an isotonic force transducer and equilibrated for at least 1 h before exposure to drugs. Acetylcholine was used for muscle stimulation. RESULTS: Thienorphine (0.005-1.0 mg/kg, ig) or buprenorphine (0.005-1.0 mg/kg, sc) dose-dependently significantly inhibited gut transit compared with saline. Thienorphine inhibited gut transit less than buprenorphine. The maximum inhibition by thienorphine on the intestinal transit was 50%-60%, whereas the maximum inhibition by morphine on gut transit was about 100%. Thienorphine also exhibited less inhibition on acetylcholine-induced contraction of GPI, with a maximum inhibition of 65%, compared with 93% inhibition by buprenorphine and 100% inhibition by morphine. Thienorphine induced a concentration-dependent decrease in the basal tonus of spontaneous movement of the GPI, the effect of which was weaker than that with buprenorphine. The duration of the effect of thienorphine on the GPI was longer than that with buprenorphine. CONCLUSION: Thienorphine had less influence, but a longer duration of action on GPI contraction and moderately inhibited intestinal transit.

[Primary investigation on heterodimerization of kappa-opioid receptor and ORL1 receptor].

This study investigates whether kappa-opioid receptor and ORL1 receptor may interact to form a heterodimer. In immunofluorescence and co-immunoprecipitation experiments, differentially epitope-tagged receptors, colocalization and heterodimerization of kappa-opioid receptor and ORL1 receptor were used and examined in primary culturing rat neurons, Chinese hamster ovary (CHO) or human embryonic kidney 293 (HEK293) cells. The results show that fluorescence of both kappa-opioid receptor and ORL1 receptor were overlapping in primary culturing hippocampal and cortical neurons. Similarly in co-expressing CHO or HEK293 cells, HA-KOR and Myc-ORL1 were almost exclusively confined to the membranes, revealing extensive colocalization. When Flag-KOR and Myc-ORL1 were co-expressing in CHO cells, heterodimerization was identified to have the ability to co-immunoprecipitate ORL1-receptors with kappa-opioid receptor and vice versa. In the current study, further evidence was provided for the direct interaction of two subtypes of opioid receptors, kappa-opioid receptor and ORL1-receptor, to form the heterodimerization. The finding represents the novel pharmacological mechanism for modulation of opioid receptor function as well as diversity of G protein-coupled receptors.

Pharmacological mechanisms underlying the antinociceptive and tolerance effects of the 6,14-bridged oripavine compound 030418.

AIM: To investigate possible pharmacological mechanisms underlying the antinociceptive effect of and tolerance to N-methyl-7α-[(R)-1-hydroxy-1-methyl-3-(thien-3-yl)-propyl]-6,14-endo-ethanotetrahydronororipavine (030418), a derivative of thienorphine. METHODS: The binding affinity and efficacy of 030418 were determined using receptor binding and guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) assays in CHO-µ, CHO-κ, CHO-δ, and CHO-ORL1 cell membranes. The analgesic activity of and tolerance to 030418 were evaluated in thermal nociceptive tests in mice. The effects of 030418 on opioid receptors were further investigated using in vivo pharmacological antagonist blockade and in vitro tissue preparations. RESULTS: The compound 030418 displayed high binding affinity to all subtypes of opioid receptors with K(i) values in the nanomolar range. In [(35)S]GTPγS binding assay, the maximal stimulation of 030418 to µ-, κ-, δ-receptors and the ORL1 receptor was 89%, 86%, 67% and 91%, respectively. In hot-plate test, the antinociceptive effect of 030418 was more potent and longer than morphine. The nonselective opioid receptor antagonist naloxone could completely block 030418-induced antinociception, while both the µ-opioid receptor antagonist ß-FNA and the κ-opioid receptor antagonist nor-BNI attenuated 030418-induced antinociception. In contrast, the ORL1 receptor antagonist J-113397 enhanced the antinociceptive effect of 030418. Additionally, chronic treatment with 030418 resulted in a dramatic development of tolerance that could not be effectively prevented by J-113397. In guinea pig ileum preparation, the existing action of 030418 could be removed with difficulty after prolonged washing. CONCLUSION: The compound 030418 is a novel agonist of opioid receptors with high efficiency, long-lasting effect and liability to tolerance, which may be closely correlated with the methyl group at the N(17) position and the high hydrophobicity of the C(7)-thiophene group in its chemical structure.

TH-030418: a potent long-acting opioid analgesic with low dependence liability.

Numerous efforts have been made on the chemical modification of opioid compounds, with the ultimate goal of developing new opioid analgesics that is highly potent and low/non-addictive. In a search for such compounds, TH-030418 [7α-[(R)-1-hydroxy-1-methyl-3-(thien-3-yl)-propyl]-6,14-endo-ethanotetrahydrooripavine] was synthesized. Here, we evaluated the pharmacological activities of TH-030418, in comparison with morphine, the prototype opioid analgesic. In radioligand binding assays, TH-030418 bound potently and nonselectively to µ-, δ-, κ-, and ORL1 (opioid receptor-like 1) receptors stably expressed in CHO (Chinese hamster ovary) cells with K (i) values of 0.56, 0.73, 0.60, and 1.55 nM, respectively. When administered subcutaneously, TH-030418 was much more potent than morphine in analgesia, with the ED(50) values of 1.37 µg/kg and 1.70 µg/kg in hot plate and acetic acid writhing tests, respectively. The opioid antagonist naloxone blocked the antinociceptive effect of TH-030418, indicating that the action of TH-030418 was mediated by opioid receptors. The antinociceptive effect of s.c. TH-030418 in hot plate test lasted for more than 12 h, which is much longer than those of morphine (2.5 h) and dihydroetorphine (1.5 h). In addition, naloxone did not precipitate withdrawal syndrome in the mice treated with TH-030418 previously. Most importantly, TH-030418 did not induce conditioned place preference in mice after chronic treatment. These results indicate that TH-030418 is a potent long-acting opioid analgesic with low dependence liability and may be of some value in the development of new analgesics.

Inhibition of endothelin-1 and hypoxia-induced pulmonary pressor responses in the rat by a novel selective endothelin-A receptor antagonist, di-n-butylaminocarbamyl-L-leucyl-D-tryptophanyl-D-4-chloro-Phe.

Pulmonary hypertension is a kind of disease associated with a very high rate of mortality, and there are not many effective drugs for the treatment. Today, endothelin (ET)-1 receptor antagonists were proved to be effective in the treatment of pulmonary hypertension. Aiming at developing new endothelin-A receptor (ETA) antagonist for treatment of pulmonary hypertension, di-n-butylaminocarbamyl-L-leucyl-D-tryptophanyl-D-4-chloro-Phe, named GF063, was synthesized at base of selective ETA receptor antagonist BQ485 and selected for the further pharmacological characterization. The preliminary pharmacodynamics of GF063 was evaluated by radioligand receptor binding assay and test of antivasoconstriction effects in vitro and in vivo. The integrative pharmacodynamics was evaluated in hypoxia-induced rat pulmonary hypertension. In vitro, GF063 bound to ETA receptor with 100,000-fold higher affinity than to ETB receptor. GF063 concentration dependently inhibited contraction of isolated rat aortic ring induced by ET-1 and shifted the cumulative concentration-contraction response curve to right with no change in the maximal response. In vivo, GF063 inhibited the increase of mean systemic arterial pressure induced by ET-1 in anesthetized rat. In hypoxia-induced rat pulmonary hypertension model, pretreatment with GF063 (40 mg/kg, s.c.) significantly decreased pulmonary artery pressure and right ventricular hypertrophy, also significantly inhibited the increase of ET-1 level in lung, improved hemodynamics, and alleviated the wall thickness of pulmonary vessels. This study indicated that GF063, as a selective ETA receptor antagonist, could inhibit vasoconstriction effects in vivo and in vitro, could prevent pulmonary hypertension induced by hypoxia, and may have great potential to be developed as a new drug of antipulmonary hypertension.

Spinal antinociception of synthetic omega-conotoxin SO-3, a selective N-type neuronal voltage-sensitive calcium channel blocker, and its effects on morphine analgesia in chemical stimulus tests in rodent.

SO-3, a novel Omega-superfamily conotoxin derived from Conus striatus, selectively inhibits N-type neuronal voltage-sensitive calcium channels. In current study, antinociception of SO-3 compared with MVIIA or morphine and its effects on morphine analgesia were investigated in rodent chemical stimulus tests after acute or repeated intrathecal administration. In mice acetic acid writhing test, similar to MVIIA, SO-3 caused dose- and time-dependent spinal antinociception with ED(50) of 0.25 microg/kg and t(1/2) of 4h, which was more potent and longer-acting than morphine. In rat formalin test after intrathecal bolus injection, SO-3 produced dose- and time-dependent antinociception by suppressing acute (ED(50), 1.79 microg/kg) and tonic phases (ED(50), 0.41 microg/kg), which was similar to MVIIA and approximately 10-fold potency and twice longer-acting of morphine in blocking tonic phase responses. After repeated intrathecal injections twice daily for 5 consecutive days, SO-3 produced analgesia without loss of potency whereas morphine produced analgesia tolerance in rat formalin test; further, SO-3 still produced potent analgesia in morphine-tolerant rats. SO-3 co-administered with morphine left-shift the dose-response curve of morphine in mice acetic acid writhing test and significantly potentiated morphine analgesia in rat formalin test. No changes in motor function were seen in mice or rats receiving antinociceptive doses of SO-3 whereas MVIIA caused motor dysfunction at doses of 1.0-2.0 microg/kg in rats. This study showed that (1) novel SO-3 produced potent and long-acting spinal antinociception without observable motor dysfunction, (2) SO-3 significantly potentiated morphine analgesia, (3) After repeated intrathecal administration, SO-3 produced neither tolerance nor cross-tolerance to morphine analgesia.

[Pharmacokinetics of 6beta-naltrexol after single and multiple intramuscular injections in Beagle dogs].

The pharmacokinetics of 6beta-naltrexol (6beta-NOL) following single intramuscular administration and multiple intramuscular injection once per day for seven days was studied in 4 Beagle dogs. Plasma concentration of 6beta-NOL in dogs was analyzed by a combination of high performance liquid chromatography (HPLC) and electrochemical detection with naloxone (NLX) as internal standard. After single intramuscular injection of 0.2 mg x kg(-1) 6beta-NOL, the plasma concentration-time curve of the drug was found to fit to a two compartment model with first-order absorption. The main parameters of single dosing were as follows: t1/2alpha was (0.26 +/- 0.23) h, t1/2beta was (4.77 +/- 1.65) h, C(max) was (81.65 +/- 5.61) ng x mL(-1), t(peak) was (0.27 +/- 0.07) h, CL(s) was (1.20 +/- 0.06) L x kg(-1) x h(-1), V/F(c) was (1.94 +/- 0.15) L x kg(-1), and AUC(0-t) was (166.82 +/- 7.68) ng x h x mL(-1), separately. After multiple intramuscular injection of 0.2 mg x kg(-1) 6beta-NOL once per day for seven days, the plasma concentration-time curve of the drug fitted to a two compartment model with first-order absorption too. The main parameters of the last dosing were as follows: t1/2alpha was (0.19 +/- 0.18) h, t1/2beta was (5.79 +/- 1.50) h, C(max) was (79.82 +/- 10.5) ng x mL(-1), t(peak) was (0.18 +/- 0.08) h, CL(s) was (1.12 +/- 0.07) L x kg(-1) x h(-1), V/F(c) was (2.10 +/- 0.27) L x kg(-1), and AUC(0-t) was (173.23 +/- 9.49) ng x h x mL(-1), separately. The difference of the parameters between the first and the last dosing was not significant, showing that the plasma kinetics of 6beta-naltrexol was not changed after multiple administrations. In the course of multiple administration, the peak and valley concentration of plasma 6beta-naltrexol were (79.03 +/- 10.3) and (1.50 +/- 0.93) ng x mL(-1), respectively. No clear adverse events were noted during this study. These results showed that plasma 6beta-naltrexol fits to a two compartment model with first-order absorption in dog after intramuscular administration and their pharmacokinetic parameters were reported. There was no remarkable change on plasma pharmacokinetics of 6beta-naltrexol after multiple intramuscular administrations.

[Structure-activity relationships analysis of thienorphine and its derivatives].

Thienorphine is a chemically-new opioid developed in Beijing Institute of Pharmacology and Toxicology. To elucidate the chemical basis for the unique pharmacological effects of thienorphine, 15 derivatives were synthesized according to combinatorial chemistry and the structure-activity relationships of these compounds were studied. It is demonstrated that thienorphine is a potent long-acting partial agonist. N-Cyclopropylmethyl is responsible for the antagonist effect of thienorphine. More importantly, thiophene at the end of side chain is most likely the pharmacophore accounts for the long-lasting effect of thienorphine. Change of the connection of thiophene and the side chain does not result in changes in the antinociceptive activity.

Levo-tetrahydropalmatine attenuates oxycodone-induced conditioned place preference in rats.

Levo-tetrahydropalmatine (l-THP) is an alkaloid purified from the Chinese herb corydalis and stephania and is contained in many traditional Chinese herbal preparations. Our previous studies demonstrated the ability of l-THP to inhibit locomotor stimulation and physical dependence induced by oxycodone in mice and rats. The present study was designed to evaluate effects of l-THP on reward of oxycodone using conditioned place preference assay. Oxycodone (0.32-5.0 mg/kg) induced the development of conditioned place preference in rats. Furthermore, oxycodone (2.5 mg/kg) induced the increased phosphorylation of CREB and ERK in nucleus accumbens and hippocampus, but not in prefrontal cortex. l-THP (6.25-18.50 mg/kg) per se was not able to induce conditioned place preference or conditioned place aversion. l-THP co-administered with oxycodone during the conditioning sessions partly abolished the development of oxycodone-induced conditioned place preference in rats. Furthermore, l-THP inhibited the increased phosphorylation of ERK and CREB in nucleus accumbens and hippocampus of rats. All these results suggest that l-THP can inhibit oxycodone-induced psychological dependence by affecting phosphorylation of CREB and ERK in nucleus accumbens and hippocampus of rats. Together, the present data, combined with previous finding, support the potential use of l-THP for treatment of oxycodone addiction.

Effects of l-tetrahydropalmatine on locomotor sensitization to oxycodone in mice.

AIM: Recent studies have shown that l-tetrahydropalmatine (l-THP), an active component of Corydolis yanhusuo, can inhibit the development of the conditional place preference induced by opioid receptor agonists, but the effects of l-THP on locomotor sensitivity induced by opioid receptor agonists have not been documented. In the present study, the effects of l-THP on locomotor sensitization to oxycodone, which is an opioid receptor agonist, were studied. METHODS: Mice treated daily for 7 d with 5 mg/kg oxycodone and challenged with the same dose after 5 days of washout showed locomotor sensitization. In order to study the effects of l-THP on locomotor sensitization induced by oxycodone, l-THP was administered at doses of 6.25, 12.5, and 18.75 mg/kg, 40 min prior to treatment of oxycodone. RESULTS: l-THP per se did not affect the locomotor activity at the doses of 6.25, 12.5, and 18.75 mg/kg, but could antagonize the hyperactivity induced by oxycodone (5 mg/kg). Co-administration of l-THP (18.75 mg/kg), 40 min prior to oxycodone, could inhibit the development of sensitization to oxycodone. In addition, l-THP (6.25, 12.5, and 18.75 mg/kg, i.g.) dose-dependently prevented the expression of oxycodone sensitization. CONCLUSION: These results suggested that l-THP could attenuate the locomotor-stimulating effects of oxycodone and inhibit the development and expression of oxycodone behavioral sensitization.

[Detection of blood dissemination during the operation of lung cancer and its significance].

OBJECTIVE: To evaluate the possibility of dissemination of lung cancer cells through blood during the operation for lung cancer. METHODS: The blood samples were taken from 52 patients with non-small cell lung cancer (NSCLC) and 5 patients with benign lung diseases at four different intervals during the operation. The transcription of carcinoembryonic antigen (CEA) messenger ribonucleic acid was assayed by means of nested reverse transcriptase polymerase chain reaction (RT-PCR). A549 (a human adenocarcinoma cell line) served as positive control. The sensitivity has been tested using quantificationally diluted A549 cells. RESULTS: The CEA mRNA positive rates of all four time spots are as follows: 31% (16/52) at beginning of the operation (sample taken from peripheral vein), 54% (28/52) at ligating the pulmonary vein (peripheral vein), 54% (28/52) at ligating the pulmonary vein (pulmonary vein) and 54% (28/52) at 1 hour after ligating the pulmonary vein (peripheral vein). There is no relationship between the tumor identity and the positive rate of CEA mRNA. The positive rate of CEA mRNA is higher in patients with centrally located lung cancer than that in patients with peripherally located lung cancer, similar phenomenon is also found between patients with advanced lung cancer and the patients with early stage of lung cancer. No negative control samples was found to be positive for CEA mRNA, the sensitivity of our test was 1 x 10(-6). CONCLUSIONS: The cancer cell dissemination during operation was demonstrated indirectly in our study, the time of pulmonary vein ligation (later or earlier) may affect the quantity of tumor cells released into circulation. Patients with lung cancer of central type and late TNM stage have more possibility of cancer cell dissemination during operation. More effective means may be needed to avoid the dissemination of cancer cells.

[Comparison of the antagonistic effects of 6 beta-naltrexol and naltrexone against morphine analgesia].

AIM: To compare the antagonistic effects of 6 beta-naltrexol and naltrexone against morphine analgesia. METHODS: The effects of 6 beta-naltrexol and naltrexone against morphine analgesia were observed in mouse heat radiant tail-flick assay and in mouse (55 +/- 1) degrees C hot plate test. The displacement of 6 beta-naltrexol and naltrexone on binding to CHO-mu receptor was observed by radioligand binding study. RESULTS: 6 beta-naltrexol antagonized morphine analgesia but the potency was (6.1 +/- 1.7)% that of naltrexone. The effective duration of 6 beta-naltrexol was 3-4 times that of naltrexone and the peak time of the response was about 0.5-1 h after s.c. equivalent efficacy dose (ED95) in two models. Like naltrexone, 6 beta-naltrexol was effective by oral administration and the potency ratio of p.o./s.c. was 1/3. As an antagonist to opioid receptor, the displacement of 6 beta-naltrexol was about 12.5% that of naltrexone, which was almost in agreement with the efficacies against morphine analgesia in mouse. CONCLUSION: Compared with naltrexone, 6 beta-naltrexol was less potent but duration was longer.

[Significance of microvascular density and vascular endothelial growth factor in breast cancer].

OBJECTIVE: To study the significance of vascular endothelial growth factor (VEGF) and microvascular density (MVD) expression in breast cancer. METHODS: The expression of MVD and VEGF was studied in 81 patients with primary breast cancer by SP immunohistochemical technique. RESULTS: There were 49 patients with high VEGF expression (60.5%) and 35 patients with high MVD expression (43.2%). There was significant correlation between VEGF or MVD expression and TNM stage, but not age, tumor size, ER expression or lymph node status. VEGF was significantly related with MVD expression. Univariate analysis showed that patients with low expression of VEGF and MVD had longer disease free survival (DFS) and overall survival (OS). Grouping univariate analysis showed that VEGF had significant correlation with the DFS of all grouped patients and the OS of patients with LN(+) or stage III lesions. MVD had significant correlation with the DFS and OS of LN(+) patients and the DFS of stage III lesion patients. Multivariate analysis showed that MVD was a risk predictor because of its positive statistic value, the higher expression, the shorter survival time of the patients. CONCLUSION: Both VEGF and MVD are useful prognostic factors in breast cancer. MVD appears to be a strong and independent biologic marker for breast cancer prognosis.