Up-regulation of HLA-G expression in cervical premalignant and malignant lesions.

The human leukocyte antigen G (HLA-G) molecule, a non-classical major histocompatibility complex class I antigen, exhibits highly limited tissue distribution and gene variation. Recent studies indicate strong immunoinhibitory properties in tumor cells that may favor their escape from anti-tumor immune responses. However, the role of HLA-G in cervical premalignant and malignant lesions has not been defined clearly. In our study, HLA-G expression was studied in cervical tissue from 119 patients with lesions and 22 normal cervical tissue specimens by immunohistochemistry. HLA-G was expressed in 45% (54/119) of cervical lesion-containing tissues while it was rarely detectable (0/22) in the control specimens (P = 0.000). ROC curve analysis showed that HLA-G has an area under the curve (AUC) of 0.694. Furthermore, we investigated soluble HLA-G expression in the plasma of 172 patients with cervical lesions and 20 healthy controls. Significant increases were also observed in soluble HLA-G levels (median, 191.4 vs 45.18 U/ml, P < 0.001). The relative operating characteristic (ROC) curves for soluble HLA-G (sHLA-G), squamous cell carcinoma (SCC), and carbohydrate antigen 125 (CA125) show an AUC of 0.710, 0.634, and 0.588, respectively. At the cut-off values of 108.20 U/ml for sHLA-G, 1.5 ng/ml for SCC, and 35 U/ml for CA125, the sensitivity was 73.30%, 47.83%, and 44.83%, respectively. The detection of soluble HLA-G in plasma may have significance in the early detection of cervical malignant lesions.

Targeted hepatocellular carcinoma proapoptotic BikDD gene therapy.

Hepatocellular carcinoma (HCC), the third leading cause of cancer death in the world, is the most general type of primary liver cancer. Although current treatment modalities, such as liver transplantation, resection, percutaneous ablation, transarterial embolization, chemotherapy and radiotherapy are potentially curative, these methods are not universally applicable to all of HCC patients, especially for those with poor prognosis in which no effective remedy is available. Therefore, development of novel therapeutic approach for the treatment of HCC is urgently needed. In the current study, we developed a promising HCC-targeted gene therapy vector driven by liver cancer-specific α-fetoprotein promoter/enhancer coupled to an established platform technology. The activity of this expression vector is comparable with or even higher than that of strong cytomegalovirus (CMV) promoter and exhibits strong promoter activity in liver cancer cells/tumors, but has nearly no or very low activity in normal cells/organs in vitro and in orthotopic animal models in vivo. Its cancer specificity exceeds that of the CMV promoter, which expresses non-specifically in both normal and tumor cells. In addition, targeted expression of a therapeutic BikDD, a mutant of proapoptotic gene Bik effectively and preferentially killed liver cancer cells, but not normal cells and significantly repressed growth of HCC tumors, and prolonged survival in multiple xenograft and syngeneic orthotopic mouse models of HCC through intravenous systemic gene delivery. Importantly, systemic administration of BikDD by our expression vector exerted no systemically acute toxicity compared with CMV-BikDD in mice. Taken together, this study elucidates a relatively safe and highly effective and specific systemic gene therapy strategy for liver cancer, and is worthy of further development for future clinical trials.

Cancer targeted gene therapy of BikDD inhibits orthotopic lung cancer growth and improves long-term survival.

Lung cancer is a leading cause of cancer death due to the high incidence of metastasis; therefore, novel and effective treatments are urgently needed. A current strategy is cancer-specific targeted gene therapy. Although many identified that cancer-specific promoters are highly specific, they tend to have low activity compared with the ubiquitous cytomegalovirus (CMV) promoter, limiting their application. We developed a targeted gene therapy expression system for lung cancer that is highly specific with strong activity. Our expression vector uses the survivin promoter, highly expressed in many cancers but not normal adult tissues. We enhanced the survivin promoter activity comparable to the CMV promoter in lung cancer cell lines using an established platform technology, whereas the survivin promoter remained weak in normal cells. In mouse models, the transgene was specifically expressed in the lung tumor tissue, compared with the CMV promoter that was expressed in both normal and tumor tissues. In addition, the therapeutic gene BikDD, a mutant form of pro-apoptotic Bcl2 interacting killer, induced cell killing in vitro, and inhibited cell growth and prolonged mouse survival in vivo. Importantly, there was virtually no toxicity when BikDD was expressed with our expression system. Thus, the current report provides a therapeutic efficacy and safe strategy worthy of development in clinical trials treating lung cancer.

Inhibitory effects of digitalis on the proliferation of androgen dependent and independent prostate cancer cells.

PURPOSE: Digitalis or cardiac glycosides have been noted to induce tumor static or oncolytic effects in various types of cancer. We evaluated the effects and underlying mechanisms of cardiac glycosides, including digoxin, digitoxin and ouabain, on the proliferation of hormone dependent and independent prostate cancer cell lines. MATERIALS AND METHODS: Cell proliferation of the 3 human prostate cancer cell lines LNCaP, DU145 and PC3 was measured by 3-(4,5-dimethylthiazol-2-yle)2,5-diphenyltetralozium bromide (Sigma Chemical Co., St. Louis, Missouri) colorimetric assay. The cytotoxic effects of digitalis on prostate cancer cells were determined by lactate dehydrogenase measurements of the culture medium. Intracellular Ca2+ was measured by a dual wavelength spectrometer system. The percent of apoptotic cells after digitalis treatment was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling and flow cytometry. RESULTS: Digoxin, digitoxin and ouabain significantly inhibited the proliferation of LNCaP, DU145 and PC3 cells at a dose of 1 or 10 microM. after 1 to 4 days of culture. Cytotoxicity of digitalis on the DU145 and LNCaP cells was dose dependent but cytotoxicity was not obvious in PC3. Digitalis (1 microM.) significantly increased intracellular Ca2+ in LNCaP and DU145 after 12 hours of culture but PC3 cells needed a 24-hour treatment to show any effect. In the apoptosis measurement digitalis at a dose of 1 and 10 microM. also significantly increased the percent of apoptotic cells in the LNCaP, DU145 and PC3 cell lines. Normal control human glomerular epithelial cells showed no response to digitalis treatment at all tested doses. CONCLUSIONS: Digitalis may inhibit the proliferation of prostate cancer cell lines, although the 3 cell lines showed varied sensitivity to digitalis. These effects are possibly the result of a mechanism involving sustained elevation of the concentration of intracellular Ca2+ and of apoptosis.

Effects of hyperprolactinemia on testosterone production in rat Leydig cells.

The pathogenesis of hyperprolactinemia (hyperPRL) induced hypogonadism has been suggested to be related with a dysfunction of hypothalamus-pituitary-testis axis. While the direct inhibitory effects of prolactin (PRL) on testosterone (T) release have been demonstrated, the mechanism is still unclear. Our previous study demonstrated a diminished T release in the testicular interstitial cells (TICs) from the anterior pituitary (AP)-grafted rats as compared with the control, and the pattern was in agreement with the in vivo model. However, TICs incubation cannot totally represent the response of the Leydig cells. Therefore, a Percoll gradient purified Leydig cell model was adopted to explore the response of T release under similar challenges in this study to investigate the effects of hyperPRL on the Leydig cells per se. HyperPRL in male rats was induced by grafting rat AP under the renal capsule. The control animals were grafted with rat brain cortex tissue (CX). Six weeks after grafting, the rats were sacrificed. Either TICs or Leydig cells were isolated, respectively, for in vitro incubation and challenge. Challenge drugs included human chorionic gonadotropin (hCG, 0.05 IU/ml), steroidogenic precursors (25-OH-cholesterol, 10(-6) M; pregnenolone, 10(-6) M), forskolin (an anenylyl cyclase activator, 10(-4) M) and 8-bromo-3':5' cyclic adenosine monophosphate (cAMP) (8-Br-cAMP 10(-4) M). T released by TICs or Leydig cells was determined by radioimmunoassay. The TICs from the AP-grafted rats showed lower levels of T release than the control group while the purified Leydig cells demonstrated a reverse pattern in response to challenges of hCG, steroidogenic precursors, forskolin and 8-Br-cAMP. In hyperPRL rats, a paradoxical pattern of T release between TICs and purified Leydig cells is observed. The purified Leydig cells from AP-grafted rats demonstrated a higher level amount of T release than the control after stimulation. The phenomenon can be attributed to the change of Leydig cell sensitivity to the stimulation after the effects of chronic hyperPRL. Moreover, another possibility is the role played by other interstitial cells to modulate steroidogenesis in Leydig cells.

Effects of fasting on aldosterone secretion in ovariectomized rats.

The present study was designed to assess the effect of fasting on aldosterone secretion in ovariectomized (Ovx) rats. Ovx rats were divided into fed (allowed access to food ad libitum) and fasted (deprived of food for 24 hours) groups. The trunk blood of fed and fasted rats was collected after decapitation. In the in vitro study, adrenal zona glomerulosa (ZG) cells from fed or fasted rats were incubated with angiotensin II (Ang II, 10(-6) M), adrenocorticotropic hormone (ACTH, 10(-9) M), or forskolin (an activator of adenylyl cyclase, 10(-6) M) at 37 degrees C for 30 min. The levels of aldosterone in medium and plasma extracts were measured by radioimmunoassay. Results showed that the levels of plasma aldosterone in fasted rats were lower than those in fed rats. There were no significant differences in basal and Ang II-stimulated aldosterone secretion between fed and fasted groups. The increment of aldosterone induced by ACTH in fasted group was significantly less than that in fed group. Administration of forskolin led to a significant increase in aldosterone secretion in both fed and fasted groups. Fasted group had a decreased aldosterone secretion in response to forskolin as compared with fed group. In summary, these results suggest that fasting decreases aldosterone secretion in Ovx rats through a mechanism in part involving a reduction of aldosterone production in response to ACTH, a decreased activity of adenylyl cyclase, and/or an inhibition of post-cAMP pathway in ZG cells.

Direct effects of prolactin on corticosterone release by zona fasciculata-reticularis cells from male rats.

The role of prolactin (PRL) in the male is not fully defined. The aim of this study was to investigate the function and mechanism of PRL on the production of corticosterone by zona fasciculata-reticularis (ZFR) cells in vitro. The ZFR cells were obtained from male rats under normal, hyperprolactinemic, or hypoprolactinemic situation. PRL stimulated the corticosterone release in a dose-dependent pattern in the ZFR cells from normal male rats. The cellular adenosine 3'-5'-cyclic monophosphate (cAMP) concentration positively correlated with PRL concentration in the presence of forskolin or 3-isobutyl-1-methylxanthine (IBMX). PRL enhanced the stimulatory effects of cAMP mimetic reagents, i.e., forskolin, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP), and IBMX on the release of corticosterone. The adenylate cyclase inhibitor (SQ22536) inhibited the corticosterone release in spite of presence of PRL. Nifedipine (L-type calcium channel blocker) did not inhibit corticosterone release. The hyperprolactinemic condition was actualized by transplantation of donor rat anterior pituitary glands (APs) under kidney capsule. By comparison with the cerebral cortex (CX)-grafted group, AP-graft resulted in an increased release of corticosterone, 3beta-hydroxysteriod dehydrogenase (HSD) activity and cAMP production by ZFR cells. Acute hypoprolactinemic status was induced by bromocriptine for 2 days. The results showed the productions of corticosterone were lower in hypoprolactinemic group than in control group, which were persistent along with different ACTH concentrations. These results suggest that PRL increase the release of corticosterone by ZFR cells via cAMP cascades and 3beta-HSD activity.