[Clinical analysis of 140 cases of mantle cell lymphoma].

Objective: To study the clinical features, therapeutic effects, prognostic factors of 140 patents with mantle cell lymphoma (MCL). Methods: Clinical data of 140 MCL patients admitted from June 2009 to January 2016 in our hospital were retrospectively analyzed. Results: The median age of 140 patients was 59 years with a ratio of 6∶1 for men and women. There were 134 cases (95.7%) in Ann-Arbor stage Ⅲ-Ⅳ, 37 cases (26.4%) with B symptoms, 61 cases (43.6%) with bone marrow involvement and 38 cases (27.1%) with enlarged spleen. The overall response rate (ORR), 3-year survival rate and progression-free survival rate in the treatment group with rituximab were 87.1%, 68.1% and 59.5% respectively, which were significantly higher than those in the rituximab-free treatment group (66.6%, 51.5% and 31.7%, respectively). The difference was statistically significant (all P<0.05). Among patients treated with rituximab, the complete remission rates (70.8% and 77.8%) of R-HyperCVAD/MA and VcR-CAP regimens were higher than those of R-CHOP regimen (39.0%, both P<0.05). However, there was no significant difference in the overall response rate, overall survival rate and progression-free survival rate (all P>0.05). Univariate analysis showed that age, Ki-67 index, B symptoms, bone marrow invasion, platelet count, LDH, ß(2)-MG and MIPI scores were associated with overall survival (all P<0.05). Multivariate analysis showed that age (HR=4.940, 95% CI: 2.347 to 10.397), B symptom (HR=2.900, 95% CI: 1.517-5.544), ß(2)-MG (HR=2.945, 95% CI: 1.656-5.238), Ki-67 index (HR=4.915, 95% CI: 2.554-9.456) and treatment with rituximab-containing regimen (HR=2.450, 95% CI: 1.352-4.440) were independent factors for OS. Conclusions: Most patients with MCL were older adults and usually had bone marrow involvement and spleen involvement. Rituximab combined with chemotherapy (especially R-HyprCVAD/MA and VcR-CAP) had better clinical efficacy than conventional chemotherapy.

Junctophilin 3 expresses in pancreatic beta cells and is required for glucose-stimulated insulin secretion.

It is well accepted that junctophilin (JPHs) isoforms act as a physical bridge linking plasma membrane and endoplasmic reticulum (ER) for channel crosstalk in excitable cells. Our purpose is to investigate whether JPHs are involved in the proper communication between Ca(2+) influx and subsequent Ca(2+) amplification in pancreatic beta cells, thereby participating in regulating insulin secretion. The expression of JPH isoforms was examined in human and mouse pancreatic tissues, and JPH3 expression was found in both the beta cells. In mice, knockdown of Jph3 (si-Jph3) in islets decreased glucose-stimulated insulin secretion (GSIS) accompanied by mitochondrial function impairment. Si-Jph3 lowered the insulin secretory response to Ca(2+) signaling in the presence of glucose, and reduced [Ca(2+)]c transient amplitude triggered by caffeine. Si-Jph3 also attenuated mitofusin 2 expression, thereby disturbing the spatial organization of ER-mitochondria contact in islets. These results suggest that the regulation of GSIS by the KATP channel-independent pathways is partly impaired due to decrease of JPH3 expression in mouse islets. JPH3 also binds to type 2 ryanodine receptors (RyR2) in mouse and human pancreatic tissues, which might contribute to Ca(2+) release amplification in GSIS. This study demonstrates some previously unrecognized findings in pancreatic tissues: (1) JPH3 expresses in mouse and human beta cells; (2) si-Jph3 in mouse primary islets impairs GSIS in vitro; (3) impairment in GSIS in si-Jph3 islets is due to changes in RyR2-[Ca(2+)]c transient amplitude and ER-mitochondria contact.

MicroRNA-181a-mediated downregulation of AC9 protein decreases intracellular cAMP level and inhibits ATRA-induced APL cell differentiation.

AC9 is one of the adenylate cyclase (AC) isoforms, which catalyze the conversion of ATP to cAMP, an important second messenger. We previously found that the integration of cAMP/PKA pathway with nuclear receptor-mediated signaling was required during all-trans retinoic acid (ATRA)-induced maturation of acute promyelocytic leukemia (APL) cells. Here we showed that AC9 could affect intracellular cAMP level and enhance the trans-activity of retinoic acid receptor. Knockdown of AC9 in APL cell line NB4 could obviously inhibit ATRA-induced differentiation. We also demonstrated that miR-181a could decrease AC9 expression by targeting 3'UTR of AC9 mRNA, finally controlling the production of intracellular cAMP. The expression of miR-181a itself could be inhibited by CEBPα, probably accounting for the differential expression of miR-181a in NB4 and ATRA-resistant NB4-R1 cells. Moreover, we found that AC9 expression was relatively lower in newly diagnosed or relapsed APL patients than in both complete remission and non-leukemia cases, closely correlating with the leukemogenesis of APL. Taken together, our studies revealed for the first time the importance of miR-181a-mediated AC9 downregulation in APL. We also suggested the potential value of AC9 as a biomarker in the clinical diagnosis and treatment of leukemia.

Kinetics of aristolochic acid I after oral administration of Radix Aristolochiae or Guanxinsuhe preparation in canines.

ETHNOPHARMACOLOGICAL RELEVANCE: Aristolochic acid I (AAI), a major component derived from Aristolochia species, which have been known for a long time and remain in use today, particularly in Asia and Central America. It has been confirmed to induce a type of so-called aristolochic acid nephropathy (AAN) and involved in the development of Balkan endemic nephropathy (BEN). AIM OF THE STUDY: To investigate the kinetic of AAI in beagle dogs after single-dose oral administration of Radix Aristolochiae or its preparation, Guanxinsuhe, as well as the effects of compound compatibility in traditional Chinese medicine on the pathologic processes of AAN. MATERIALS AND METHODS: Beagle dogs were orally administrated Radix Aristolochiae (0.3 g/kg/day), Guanxinsuhe preparation (0.9 g/kg/day) (with an identical dosage of AAI), and empty capsules respectively for 180 days. Canines (n=2) were euthanized on day 90, 180, 210, HPLC was established to determine the AAI level in plasma and the kinetic behaviors of AAI in dogs were elucidated after single dosing. Hematoxylin and eosin (H&E)-staining was applied for histopathologic examination to evaluate the pathological status of kidneys. RESULTS: Compared to canines with Radix Aristolochiae treatment, the Cmax, AUC, Tmax, and t(1/2ß) of AAI in Guanxinsuhe preparation group were elevated, while t(1/2α) of AAI was decreased. The results indicated the co-existing components in Guanxinsuhe preparation could increase the absorption, accelerate the distribution, but delay the absorption and elimination of AAI. After long-term dosing, animals treated with Radix Aristolochiae were found with more severe renal impairment and higher AAI level in plasma. CONCLUSIONS: It was demonstrated that the compound compatibility in Guanxinsuhe preparation can affect the kinetic process of AAI and attenuate the toxic effect on kidney when the duration of treatment was prolonged.

Adrenomedullin enhances baroreceptor reflex response via cAMP/PKA signaling in nucleus tractus solitarii of rats.

Adrenomedullin (ADM), a 52-amino acid peptide, elicits differential cardiovascular responses when it is administered systemically or directly to the brain. We evaluated in the present study the hypothesis that ADM may modulate baroreceptor reflex (BRR) response through an ADM receptor-mediated cAMP/ protein kinase A (PKA)-dependent mechanism in the nucleus tractus solitarii (NTS), the terminal site for primary baroreceptor afferents, using Sprague-Dawley rats. Our immunoblot and immunohistochemical results showed that the two component proteins of the ADM(1) receptor complex, calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP)-2, were uniformly distributed and highly co-localized in the NTS. Site-specific microinjection of ADM (0.02-0.2pmol) unilaterally into the NTS significantly increased BRR response and sensitivity in a time- and dose-related manner, without affecting arterial pressure and heart rate. The BRR enhancing effect of ADM was also temporally correlated with an up-regulation of PKA(beta), the active form of PKA and an increase in PKA activity. In addition, the ADM-evoked BRR enhancement or PKA activation was abolished by co-microinjection with a selective ADM(1) receptor antagonist, ADM(22-52), an adenylyl cyclase inhibitor, SQ22536, or a PKA inhibitor, Rp-8-bromo-cAMP. These results suggest that ADM enhances BRR via activation of a cAMP/PKA-dependent mechanism by acting site-specifically on ADM(1) receptors in NTS.

Prenylation enhances cytotoxicity of apigenin and liquiritigenin in rat H4IIE hepatoma and C6 glioma cells.

Antioxidative as well as cytotoxic effects of the prenylated flavonoids licoflavone C (8-prenylapigenin) and isobavachin (8-prenylliquiritigenin) were investigated in comparison to the corresponding non-prenylated flavonoids (apigenin, liquiritigenin) and vitexin (apigenin-C8-glucoside) using metabolically active H4IIE hepatoma and metabolically poorly active C6 glioma cells. None of the substances showed radical scavenging activities in the 2,2-diphenyl-1-picrylhydrazyl (DPPH)-assay nor were they effective in protection against H2O2-induced intracellular 2',7'-dichlorodihydrofluorescein (H2DCF) oxidation (fluorescent probe for oxidative stress) in H4IIE and C6 cells. When the intrinsic effects of the substances were investigated, licoflavone C and isobavachin exerted a pronounced toxicity in both H4IIE (IC50 values of 42+/-5 and 96+/-19 micromol/L) and C6 cells (IC50 values of 37+/-6 and 69+/-3 micromol/L) while the non-prenylated analogues as well as the glycosylated derivate vitexin showed almost no cytotoxic effect up to 250 micromol/L. In H4IIE cells the induction of apoptotic cell death by licoflavone C and icobavachin was detected as an activation of caspase 3/7 (6- and 3.3-fold, respectively). Based on these experiments we suggest that C8-prenylation of a flavonoid enhances the cytotoxicity inducing an apoptotic cell death in H4IIE cells without affecting antioxidative properties.

Estrogenic effects of two derivatives of icariin on human breast cancer MCF-7 cells.

The aims of the present study were to determine the estrogenic activities of icariin (ICA) and its derivatives and their structure-estrogenic activity relationship. Therefore, icaritin (ICT) and desmethylicaritin (DICT) were derived from ICA. The estrogenic activities of ICA, ICT and DICT were examined by cell proliferation and progestogen receptor mRNA expression of estrogen-receptor-positive MCF-7 cells. Current studies exhibited that ICT and DICT both markedly enhanced the proliferation of MCF-7 cells; as compared to estradiol (100%), their relative proliferative effects (RPE) were 90% and 94%, respectively. Cell proliferation induced by ICT and DICT was completely antagonized by ICI182,780. ICT and DICT increased progestogen receptor (PR) at mRNA levels at 48 h after treatment, although the effects were not as prominent as 17beta-estradiol (E2). Those phenomena were not observed with ICA. Results demonstrate that ICT and DICT (nonconjugated forms) possess estrogen-like activity; however, ICA appears to have no estrogenicity in the MCF-7 cell line model in vitro.

Effects of cyclophosphamide on protein expression of rat embryo at end of pre-gastrulation stage in vivo.

AIM: To seek a sensitive time point for pre-gastrulation embryos exposed to developmental toxic agents, and to establish a molecular biomarker to evaluate the mechanism of cyclophosphamide-induced embryonic abnormalities in vivo. METHODS: Pregnant rats on d 3 of gestation were given i.p. cyclophosphamide (Cyc) 10, 20, 40 mg.kg-1. SDS-PAGE was performed to qualitatively observe the target proteins in d 8 rat embryos. RESULTS: The expression of the protein with a molecular weight (M(r)) of approximately 70 kDa distinctively increased and that of the blastocyst-specific protein (M(r) 14.4 kDa) disappeared in Cyc 40 mg.kg-1 group. CONCLUSION: Day 8 of rat gestation could be an optimum time point for understanding developmental toxicity of mammalian embryo during pregastrulation, and the expression of the proteins with M(r) 70 kDa and 14.4 kDa at this point could be employed as a molecular biomarker to demonstrate embryoteratology objectively and sensitively.

Immunosurgical studies on cytological and cytogenetic toxicity analysis of rat blastocysts after in vivo exposure to cyclophosphamide.

AIM: To establish immunosurgery and indices of cytogenetic assessment for blastocyst and its inner cell mass (ICM), and to evaluate the toxic effects after in vivo exposure to cyclophosphamide. METHODS: Modified immunosurgery was established by preparation of rabbit-anti-rat spleen antiserum and induction of diluted rat mixed serum as complement. Pregnant rats on d 3 of gestation were injected i.p. cyclophosphamide (10, 20, and 40 mg.kg-1). On d 4, immunosurgery was performed on rat blastocysts. The cell number and the micronuclei of blastocyst and ICM were evaluated respectively. RESULTS: In the cyclophosphamide-treated rats, decreases of cell number (35 +/- 3, 32 +/- 1, 30 +/- 1, and 14 +/- 2, 11 +/- 1, 9 +/- 2) and increases of frequency of micronuclei (1.81%, 2.27%, 3.14%, and 2.53%, 2.98%, 4.75%) in blastocysts and ICM were observed in a dose-related manner. The changes of blastocyst were, however, not parallel to those of ICM which were more serious. CONCLUSION: Modified immunosurgery, an objective and elegant technique, was used on rat blastocysts. In vivo could cyclophosphamide injured ICM more than blastocysts.

[Effects of blastocyst deficiencies induced by aspirin treatment during preimplantation period in rats on development of embryos after implantation].

Pregnant rats were treated orally with aspirin 0.5 or 1.0 g.kg-1 on d 3 (positive vaginal smear was considered as d 0) and were sacrificed on d 4. Some blastocysts collected on d 4 were evaluated for gross morphology and cell number, and the remainings were transfered into pseudopregnant rats. Results showed that the rate of blastocysts with abnormal morphology were 23.8% and 40.8%, respectively, in 0.5 and 1.0 g.kg-1 of aspirin. These were significantly higher than 6.8% of the control group. The cell number of blastocysts also decreased in the aspirin groups. The rate of implantation and live fetuses in the case of blastocysts with normal morphology were related negatively with the aspirin doses, espesially in the group of 1.0 g.kg-1 of aspirin, the implantation rate was significantly lower (38.6%). However, the implantation rate of blastocysts with abnormal morphology in both groups of aspirin were much less than that of the control group, and all embryos after implantation were resorped. No significant malformations were observed in the live fetuses. These results suggests that the effects of blastocyst deficiencies induced by aspirin on development of embryos transfered into pseudopregnant rats mainly caused death of embryos, but not malformation of fetuses.

Toxicity to transferred rat embryos after aspirin treatment during preimplantation stage in vivo.

AIM: To explore the relationship between drug-induced blastopathies and post-implantation embryotoxicity or developmental defects. METHODS: Pregnant rats on d 3 were given intragastrically aspirin (0.25, 0.5, and 1 g.kg-1). On d 4, the blastocysts were transferred into the uterine horns of pseudopregnant rats (made by mating with male rats which had been given intragastrically 3-chloro-1,2-propanediol 5 mg.kg-1 for 5 d). Uterine contents were examined at term. RESULTS: The frequency of blastocysts with morphological alterations (FBMA) was increased on d 4 of gestation. The implantation rate was lower than that of the controls. A dose-related increase in resorption (55.2%, 69.5%, and 85.2%) and malformation rate (3.8%, 44.4%, and 25%), and decrease in viability rate of fetuses (44.8%, 30.5%, and 14.8%) were observed in test groups with correlations to FBMA. CONCLUSION: Embryotoxicity and fetal malformations were induced by treatment of aspirin before implantation in a dose-dependent manner.

[Effects of preimplantation treatment with aspirin and acetaminophen on blastocyst and fetus in rats].

Pregnant rats were treated with ig aspirin (Asp) and acetaminophen (Ace) on d 3 of pregnancy (positive vaginal smear = d 0). Blastocysts were collected on d 4 and evaluated for gross morphology, cell number, micronucleus, and mitotic index. Some rats were killed on d 20 and fetuses were examined for teratogenic effects. On d 4 a reduction of cell number per blastocyst was found in the rats treated with Asp 0.5, 1 g.kg-1, and Ace 1 g.kg-1, while the mitotic index, frequency of micronuclei, and frequency of blastocysts with morphological alterations were increased. The frequency of micronuclei was increased in rats exposed to Ace 0.25 and 0.5 g.kg-1. On d 20 major malformation and embryotoxicity were seen in Asp 0.5, 1, and Ace 1 g.kg-1 groups.