Jianliu Wang

Rapid translocation and pharmacokinetics of hydroxylated single-walled carbon nanotubes in mice

Determining the in vivo pharmacological profiles of carbon nanotubes (CNTs) is essential for the promising biomedical applications of CNTs, such as drug delivery. Using iodine-131 tracing we studied the fundamental behavior of hydroxylated single-walled CNTs (SWNTols) shortly after they were introduced into the animal body (from 2 min to 1 h) by providing the biodistribution data and pharmacokinetic parameters. The distribution was slightly influenced by injection modes, but in any mode radioactivity was found all over the body within 2 min except brain. Liver, kidneys, stomach and lungs are the target organs with high uptake of SWNTols. The SWNTols content in several tissues, such as heart, lungs, and muscle is positively correlated with its content in the blood, showing clearly that the blood stream brings SWNTols to the whole body. This work presents the initial in vivo behavior of the water-soluble functionalized SWNTs, providing also the basic data to show opportunities and limitations for realization of the CNT-based drug vehicle.

Nongenomic effect of estrogen on the MAPK signaling pathway and calcium influx in endometrial carcinoma cells

17β‐Estradiol (E2) is well known to interact with intracellular receptors that act as nuclear transcription factors. However, abundant evidence now indicates that E2 can also rapidly induce several nongenomic effects through signaling pathways related to cell growth, preservation, and differentiation. We studied the nongenomic effects of E2 in two human endometrial carcinoma cell lines, Ishikawa (estrogen receptor (ER) positive) and Hec‐1A (ER negative or low) by cultivating them with either E2 or its membrane‐impermeable conjugate, E2–BSA. We found that phosphorylation of Erk1/2 could be induced by either E2 or E2–BSA in Ishikawa cells. In Hec‐1A cells, only E2 was able to induce Erk1/2 phosphorylation. Although the existence of a nongenomic component to the response was indicated by the finding that it could not be completely inhibited by the ER antagonist ICI182780,and it can also be inhibited by calcium inhibitor Nifedipine partly. Phosphorylation of Akt could not be induced, either by E2 or E2–BSA, in either cell line. Both E2 and E2–BSA elicited calcium influx in Ishikawa cells. In contrast to these nongenomic effects, only E2 was able to stimulate expression of the anti‐apoptotic‐protein Bcl‐2. Taken together, these data indicate that nongenomic effects such as Erk1/2 phosphorylation and calcium influx can be initiated from the membrane in Ishikawa cell, and calcium can activate Erk1/2 phosphorylation. Except for ER, there must be other binding location of estrogen in endometrial cancer cells, and the nongenomic effects of estrogen initiated from plasma membrane by E2–BSA cannot lead to transcriptional effect of Bcl‐2 expression. J. Cell. Biochem. 106: 553–562, 2009.

Overexpression of the Insulin Receptor Isoform A Promotes Endometrial Carcinoma Cell Growth

Epidemiological studies have demonstrated that type 2 diabetes mellitus (T2DM) and hyperinsulinemia are associated closely with endometrial carcinoma risk, although the molecular mechanism remains unclear. Insulin receptor isoformA expression is upregulated in many cancer cells and tissues, which suggests that IR-A-mediated signaling pathways may have important implications for cancer pathogenesis. We measured the expression of insulin receptor isoforms (IR-A and IR–B in the normal endometrium tissues, the endometrial carcinoma tissues and the endometrial carcinoma cell lines. We found that the total insulin receptor (IR) and IR-A expression mRNA levels and the ratio of IR-A to total IR in endometrial carcinoma specimens were significantly higher than them in control endometrial tissue specimens(P<0.05). Further analysis indicated that the tendency was more prominently in patients with T2DM. IR-A mRNA was differentially expressed in four endometrial carcinoma cell lines (Ishikawa, KLE, RL95-2 and HEC-1-A. RL95-2 cells have a low endogenous IR-A expression, and these were used to construct a stable cell line overexpressing IR-A. We found that IR-A overexpression significantly increased cell proliferation, the proportion of cells in S phase, activation of the Akt pathway and tumorigenicity of xenografts in nude mice. In contrast, there was no significant difference in the the percentage of apoptotic cells between cells overexpressing IR-A and control cells. Moreover, levels of phosphorylated ERK1/2 protein were significantly decreased in cells overexpressing IR-A relative to controls. These findings reveal the pivotal role of IR-A in endometrial cancer carcinogenesis, and suggest that the association of elevated IR-A levels with cell proliferation and tumorigenicity may be causally linked to its effect on the proportion of cells in S phase and the activation of the Akt pathway.

Ca2+ channel subunit α 1D promotes proliferation and migration of endometrial cancer cells mediated by 17β-estradiol via the G protein-coupled estrogen receptor

Calcium and calcium channels are closely related to the estrogen‐induced nongenomic effect of endometrial carcinoma, but the specific role of calcium channels is unknown. This study aimed to explore the expression and the biologic effect of the l‐type calcium channel in endometrial carcinoma cells and to clarify the molecular mechanism of the relationship between L‐type calcium channels and estrogen. The immunohistochemical results showed that Ca2+ channel subunit α 1D (Cav1.3) expression was high in atypical hyperplasia (1.90 ± 0.35) and endometrial carcinoma tissues (2.05 ± 0.82) but weak (0.80 ± 0.15) in benign endometrial tissues (P < 0.05). Treatment with 17b‐estradiol rapidly increased Cav1.3 expression in a dose‐ and time‐dependent manner, and 100 nM cell‐impermeable β‐estradiol‐6‐(O‐carboxymethyl) oxime:bovine serum albumin also promoted Cav1.3 expression. Transfection with small interfering RNA against G protein‐coupled estrogen receptor (GPER) suppressed estrogen‐induced up‐regulation of Cav1.3 compared with control cells and markedly reduced the estrogen‐induced phosphorylation of ERK1/2 and CREB. Knocking down the Cav1.3 significantly suppressed estrogen‐stimulated Ca2+ influx, cell proliferation, and migration in endometrial cancer cells. Taken together, Cav1.3 was overexpressed in atypical hyperplasia and endometrial carcinoma, and the estrogen‐induced phosphorylation of downstream molecular ERK1/2 and CREB is the result of activation of the GPER pathway. L‐type channel Cav1.3 is required for estrogen‐stimulated Ca2+ influx and contributes broadly to the development of endometrial cancer. The Cav1.3 channel may be a new target for endometrial carcinoma treatment.—Hao, J., Bao, X., Jin, B., Wang, X., Mao, Z., Li, X., Wei, L., Shen, D., Wang, J.‐L. Ca + channel subunit a 1D promotes proliferation and migration of endometrial cancer cells mediated by 17β‐estradiol via the G protein‐coupled estrogen receptor FASEB J. 29, 2883‐2893 (2015). www.fasebj.org

The expression and role of hybrid insulin/insulin-like growth factor receptor type 1 in endometrial carcinoma cells

Insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF-IR) can assemble heteromerically as a hybrid insulin/IGF-I receptor (hybrid-R) in tissues that express both molecules. There is little information about hybrid-R in endometrial carcinoma, in which both IR and IGF-IR are frequently overexpressed. We used immunoprecipitation to detect hybrid-R expression in two endometrial carcinoma cell lines: HEC-1a, which has low estrogen receptor (ER) expression, and Ishikawa, which is positive for ER expression. To explore the role of hybrid-R in endometrial carcinoma cells, we examined phosphorylation of extracellular signal-regulated kinase (ERK1/2), which is a key molecule in the mitogen-activated protein kinase (MAPK) pathway. The effect of inhibiting IGF-I, IGF-II, and insulin on cell cycle progression and apoptosis was assessed by flow cytometry. Both cell lines expressed hybrid-R, and HEC-1a cells had higher expression levels than did Ishikawa cells. IGF-I induced ERK1/2 phosphorylation in HEC-1a cells mainly through hybrid-R; in Ishikawa cells, this effect was mediated only in part by hybrid-R. Insulin stimulated ERK1/2 phosphorylation partly through hybrid-R in HEC-1a cells, but not in Ishikawa cells. Both IGFs and insulin increased cellular DNA content in the S phase of the cell cycle in HEC-1a through hybrid-R. In contrast, in Ishikawa cells, only insulin enhanced DNA content in S phase through hybrid-R. Both IGFs and insulin significantly decreased apoptosis in HEC-1a cells through hybrid-R, and a similar but moderate effect was observed in Ishikawa cells. Hybrid-R, which is present in endometrial carcinoma cells, may have an important role in mediating IGF- and insulin-induced cell growth and in preventing apoptosis.

Supression by [D-pen2, D-pen5] enkephalin on cyclic amp dependent protein kinase-induced, but not protein kinase C-induced increment of intracellular free calcium in Ng 108-15 cells

In neuroblastoma X glioma NG108-15 cell lines, KCl 50 mM produced a significant increase in [Ca2+]i which was blocked completely by voltage-dependent Ca2+ channel antagonist verapamil. High K+-induced increase in [Ca2+i can be suppressed by selective delta opioid agonist [D-Pen2, D-Pen5] enkephalin (DPDPE) (an effect completely reversed by opioid antagonist naloxone), but not by the mu agonist ohmefentanyl (OMF) or kappa agonist 66A-078. Aside from high K+ stimulation, a number of chemicals can produce an increase in [Ca2+]i, i.e., selective adenylate cyclase activator forskolin, the membrane permeable cyclic AMP (cAMP) analogue dibutyryl-cAMP (Bt2cAMP) and the activator of protein kinase C (PKC) 12-O-tetradecanoylphorbol 13-acetate (TPA). All these effects can be readily blocked by verapamil. DPDPE blocks the increase in [Ca2+]i induced by forskolin and Bt2cAMP, but not that by TPA. The results suggest that cAMP dependent protein kinase-, but not PKC-induced Ca2+ influx mechanism seems to be involved in the delta receptor mediated opioid effect.

Mobilization of calcium from intracellular store as a possible mechanism underlying the anti-opioid effect of angiotensin II.

Angiotensin II (AII), injected intracerebroventricularly, has been shown to antagonize opioid analgesia. The mechanism for this was obscure. In the neuroblastoma X glioma NG 108-15 hybrid cell line, the K(+)-induced increase in [Ca2+]i can be suppressed by the delta opioid agonist [D-Pen2, D-Pen5]enkephalin (DPDPE) at 0.01-1 microM, an effect completely reversed by the opioid antagonist naloxone. Angiotensin II (AII) at concentrations of 0.1 and 1 microM mobilized free Ca2+ from an intracellular pool, and this effect was antagonized by the AII receptor antagonist saralasin. All (1 microM) had no significant effect on the increase in [Ca2+]i induced by K+, but it blocked the suppressive effect of DPDPE on the K(+)-induced [Ca2+]i increase. The results indicate that mobilization of intracellular calcium may underlie the anti-opioid effect of AII.

Expression of ER-α36, a Novel Variant of Estrogen Receptor in Endometrial Carcinoma and Its Clinical Significance

Objective: Estrogen receptor-α36 (ER-α36), a newly identified variant of ER-α, is predominantly membrane-based and mainly mediates nongenomic estrogen signaling. In this study, we investigated the expression of ER-α36 in human endometrial carcinoma tissues to understand the relationship between its expression and clinicopathological characteristics. Methods: ER-α36 expression was assessed by immunostaining in 73 endometrial carcinomas, 20 normal endometrial tissues, and 9 with atypical endometrial hyperplasia. Correlations between ER-α36 protein expression and clinicopathological characteristics were investigated. Results: The expression of ER-α36 in endometrial carcinoma tissues was significantly lower than in normal endometrial tissues and atypical hyperplasia (p < 0.01). ER-α36-negative tissues were significantly more likely than ER-α36-positive tumors to have tumor involvement of the cervix (p < 0.05). The disease-free survival rate of patients with ER-α36 expression was poorer than that of those who were negative for ER-α36 expression (p < 0.01). There was no significant relationship between ER-α36 expression and patient age, surgical staging, histological differentiation, myometrial invasion, lymph node metastasis, and pathological types (p > 0.05). Conclusions: ER-α36 may be an important biomarker for diagnosis, prognostication, and treatment choice in endometrial carcinoma.

The Number of Positive Pelvic Lymph Nodes and Multiple Groups of Pelvic Lymph Node Metastasis Influence Prognosis in Stage IA-IIB Cervical Squamous Cell Carcinoma

Background: Pelvic lymph node metastasis (LNM) is an important prognostic factor in cervical cancer. Cervical squamous cell carcinoma accounts for approximately 75–80% of all cervical cancers. Analyses of the effects of the number of positive lymph nodes (LNs), unilateral versus bilateral pelvic LNM and a single group versus multiple groups of pelvic LNM on survival and recurrence of cervical squamous cell carcinoma are still lacking. The study aimed to analyze the effects of the number of positive pelvic LNs and a single group versus multiple groups of pelvic LNM on survival and recurrence. Methods: We performed a retrospective review of 296 patients diagnosed with Stage IA–IIB cervical squamous cell carcinoma who received extensive/sub-extensive hysterectomy with pelvic lymphadenectomy/pelvic LN sampling at Peking University Peoples Hospital from November 2004 to July 2013. Ten clinicopathological variables were evaluated as risk factors for pelvic LNM: Age at diagnosis, gravidity, clinical stage, histological grade, tumor diameter, lymph-vascular space involvement (LVSI), depth of cervical stromal invasion, uterine invasion, parametrial invasion, and neoadjuvant chemotherapy. Results: The incidence of pelvic LNM was 20.27% (60/296 cases). Pelvic LNM (P = 0.00) was significantly correlated with recurrence. Pelvic LNM (P = 0.00), the number of positive pelvic LNs (P = 0.04) and a single group versus multiple groups of pelvic LNM (P = 0.03) had a significant influence on survival. Multivariate analysis revealed that LVSI (P = 0.00), depth of cervical stromal invasion (P = 0.00) and parametrial invasion (P = 0.03) were independently associated with pelvic LNM. Conclusions: Patients with pelvic LNM had a higher recurrence rate and poor survival outcomes. Furthermore, more than 2 positive pelvic LNs and multiple groups of pelvic LNM appeared to identify patients with worse survival outcomes in node-positive IA-IIB cervical squamous cell carcinoma. LVSI, parametrial invasion, and depth of cervical stromal invasion were identified as independent clinicopathological risk factors for pelvic LNM.

Molecular classification of human endometrial cancer based on gene expression profiles from specialized microarrays

To investigate whether the molecular classification of endometrial cancer based on gene expression profiles can predict the biological behavior of the tumors and inform prognosis.