Sorghumol triterpene inhibits the growth of circulating renal cancer cells by promoting cell apoptosis, G2/M cell cycle arrest and downregulating m-TOR/PI3K/AKT signalling pathway.

PURPOSE: To investigate the growth inhibitory effect of Sorghumol on the circulating renal cancers cells and to investigate the underlying mechanisms including its effects on apoptosis, cell cycle phase distribution and m-TOR/PI3K/AKT signalling pathway. METHODS: The antiproliferative effects were assessed by WST-1 and colony formation assay. Apoptosis was detected by the Hoechst and AO/EB staining using fluorescence microscopy. Cell cycle analysis was carried out by flow cytometry. Protein expression was checked by western blotting. RESULTS: The results revealed that Sorghumol inhibited the growth of the renal cancer cell (RCC) line A498 and circulating RCCs. However, more profound effects were observed on the RCC cells. The anticancer effects were found to be due to induction of apoptosis. Moreover, Sorghumol could also caused G2/M cell cycle arrest of the RCC cells. Besides, examination of the effect of Sorghumol on m-TOR/PI3K/AKT revealed that Sorghumol inhibited the expression of p-mTOR, p-PI3K and p-AKT in a concentration-dependent manner. CONCLUSION: Taken together, we conclude that Sorghumol inhibited the proliferation of circulating RCCs and may therefore prove to be an important lead molecule for the treatment of renal cancer.

HNF1B expression regulates ECI2 gene expression, potentially serving a role in prostate cancer progression.

Prostate cancer is the most common form of cancer in men, with increased incidence rates observed in older individuals. Prostate cancer is primarily driven via activation of the androgen receptor (AR), the principal transcriptional factor governing prostate cancer cellular programming and its associated metabolism. One of the downstream targets of AR is hepatocyte nuclear factor-1ß (HNF1B), an important oncogenic transcription factor in prostate cancer. In the present study, the regulatory role of HNF1B in enoyl-CoA-(Δ) isomerase 2 (ECI2) expression in the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model was investigated. Using this model, tumor progression and associated pathological alterations at 12, 18 and 24 weeks were analyzed. Histological sectioning revealed pathological alterations over time, including thickening of glandular epithelial cells (12 weeks), increases in cellular proliferation (18 weeks), and extensive thickening and hardening of the tissue layer (24 weeks). Expression levels of HNF1B and ECI2 proteins were validated by immunohistochemistry and western blotting at different stages of prostate cancer development. HNF1B and ECI2 exhibited minimal differences in protein expression at 12 weeks in TRAMP+ mice. However, by 18 weeks, TRAMP+ mice exhibited multi-fold increases in HNF1B expression levels, along with downregulation of ECI2. These effects were reversed at 24 weeks, indicating an important time-dependent regulation of gene expression. Taken together, these results demonstrated that upon tumor progression, the initial tumor-protective effect of HNF1B is lost along with downregulated expression of HNF1B and increased expression of ECI2.

Significant association between interleukin-10 gene polymorphisms and cervical cancer risk: a meta-analysis.

Previous studies have suggested that interleukin-10 (IL-10) polymorphisms may be associated with an increased risk of developing cervical cancer. However, the published results on this subject matter are controversial. The aim of this study was to conduct a meta-analysis of published reports to more precisely investigate the relationship between IL-10 polymorphisms and cervical cancer risk. Five online databases (PubMed, Embase, Web of SCI, CNKI and Wanfang) were searched, and seventeen articles with sufficient quantitative information were included in our meta-analysis. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the association between IL-10 polymorphisms and cervical cancer risk. Publication bias, sensitivity and cumulative analyses were also performed to support our findings. Overall, there was a significant association between the IL-10 -1082A > G polymorphism and cervical cancer risk observed in the total population (G vs. A: OR = 1.60, 95% CI = 1.12-2.29, P = 0.01, I2 = 92.3%; AG vs. AA: OR = 1.34, 95% CI = 1.04-1.74, P = 0.03, I2 = 65.9%; AG + GG vs. AA: OR = 1.58, 95% CI = 1.11-2.25, P = 0.01, I2 = 84.4%), and the same results were obtained in the subgroup analysis. Moreover, the IL-10 -819 T > C polymorphism exhibited a significant, protective effect against cervical cancer. In summary, our meta-analysis suggests that IL-10 polymorphisms may play a variety of roles in regard to cervical cancer risk, especially in Asians.

Blockage of RelB expression by gene silencing enhances the radiosensitivity of androgen­independent prostate cancer cells.

Levels of the nuclear factor­kappa B (NF­κB) alternative pathway member RelB have been shown to correlate with the effect of radiation therapy in prostate cancer. RelB expression was evaluated by immunohistochemistry in normal prostate, benign prostate hyperplasia and prostate cancer specimens. RM­1 cells were pretreated with RelB siRNA prior to radiation therapy, and RelB expression in cytoplasmic and nuclear extracts was detected by real­time polymerase chain reaction and western blot analysis. The apoptotic rates of experimental RM­1 cell groups were assessed by flow cytometry. A clonogenic growth array was used to evaluate the radiosensitivity of RM­1 cell groups. The NF­κB family member RelB was expressed at a high level in prostate cancer specimens. Compared with irradiated control cells, RM­1 cells transfected with RelB siRNA and treated with radiation therapy demonstrated a significant downregulation of RelB expression in the cytoplasm and nucleus. Notably, flow cytometry revealed that pretreatment of RM­1 cells with RelB siRNA enhanced the apoptotic rate in response to radiation therapy compared with controls. Clonogenic growth assay results revealed enhanced radiosensitivity of RelB siRNA cells at various dosage points compared with control groups. Blockage of the alternative NF­κB pathway via RelB silencing is a promising approach to enhance the radiosensitivity of prostate cancer.

[RelB-siRNA enhanced the radiosensitivity of murine prostate cancer cell line RM-1].

OBJECTIVE: To explore the transfecting effects of RelB small interfering RNA (RelB-siRNA) on murine prostate cancer cell line RM-1 sensitivity of radiotherapy. METHODS: The RM-1 cells were divided into RelB-siRNA vector, empty vector, non-transfection and normal groups. After transfection, the first three groups were irradiated by X ray. Clone formation array method was used to measure the surviving fraction and the relevant parameter values after 0, 2, 4, 6, 8 Gy irradiation. Apoptotic rate was measured via Annexin V/PI flow cytometry after 6 Gy irradiation. The level of RelBmRNA was estimated by real-time polymerase chain reaction (PCR) after 6 Gy irradiation. And the RelB protein of cytoplasm and nucleus was detected by Western blot after 6 Gy irradiation. RESULTS: Clone formation array showed that RelB group at each dose point corresponding to the survival fraction were lower that the empty vector nd non-transfection groups. The D0, Dq and SF2 values of RelB group were 1.68, 0.60, 0.43, lower than those of empty vector group 1.92, 3.08, 0.89 and non-transfection group 1.93, 2.76, 0.84. Annexin V/PI flow cytometry demonstrated that the apoptotic rate of RelB group was 15.27% ± 1.62% and it was significantly higher than the non-transfection group 7.90% ± 1.50% and the empty vector group 8.40% ± 0.69% respectively (P < 0.01) .Real-time PCR indicated that RelB-mRNA amplification of RelB group was 1.18 ± 0.03 and it was significantly lower than the non-transfection group 2.10 ± 0.61 and the empty vector group 1.97 ± 0.66 respectively (P < 0.01) .Western blot suggested that cytoplasmic gray-scale ratio of RelB group was 0.50 ± 0.08 and it was significantly lower than the non-transfection 1.77 ± 0.19 and the empty group 1.52 ± 0.12 respectively (P < 0.01) . And the nuclear gray-scale radio of the RelB group was 0.18 ± 0.03 and it was significantly lower than the non-transfection group 0.61 ± 0.12 and the empty group 0.54 ± 0.13 respectively (P < 0.01) . The RelB protein inhibition rates of cytoplasmic and nuclear RelB-siRNA were 71.8% and 70.4% respectively. CONCLUSION: RelB-siRNA can effectively inhibit the RelB expression of murine prostate cancer cell line RM-1 and significantly increase its sensitivity to radiotherapy.

Tolerogenic dendritic cells generated by RelB silencing using shRNA prevent acute rejection.

It is well known that adoptive transfer of donor-derived tolerogenic dendritic cells (DCs) helps to induce immune tolerance. RelB, one of NF-κB subunits, is a critical element involved in DC maturation. In the present study, our results showed tolerogenic DCs could be acquired via silencing RelB using small interfering RNA. Compared with imDCs, the tolerogenic DCs had more potent ability to inhibit mixed lymphocyte reaction (MLR) and down-regulate Th1 cytokines and prompt the production of Th2 cytokines. They both mediated immune tolerance via the increased of T cell apoptosis and generation of regulatory T cells. Administration of donor-derived tolerogenic DCs significantly prevented the allograft rejection and prolonged the survival time in a murine heart transplantation model. Our results demonstrate donor-derived, RelB-shRNA induced tolerogenic DCs can significantly induce immune tolerance in vitro and in vivo.

Lentiviral-mediated shRNA against RelB induces the generation of tolerogenic dendritic cells.

OBJECTIVE: Lentiviral-mediated shRNA against RelB was used to produce tolerogenic dendritic cells from murine bone marrow derived dendritic cells (BMDCs). METHOD: RelB expression in the BMDCs was silenced by lentivirus carrying RelB shRNA. The apoptosis rate and surface markers of DCs were assessed by flow cytometry. IL-12,IL-10,TGF-ß1 secreted by DCs and DNA binding capacity of NF-κB subunits in the nucleus were measured by ELISA, independently. MLR was used to analyze the capacity of DCs to inhibit immune response. RESULTS: RelB expression was significantly inhibited in DCs following lentiviral mediated delivery of RelB specific shRNA. The RelB shRNA-DC produced lower IL-12 and higher IL-10 than mature dendritic cells (mDCs) and silencing control DCs. There was no difference in the apoptosis rate between shRNA RelB-DCs and mDCs. The expression levels of co-stimulatory molecules (CD80, CD86 and CD83) and MHC-II class molecule were lower in the RelB shRNA-DCs than in the mDCs and silencing control DCs. In addition, RelB shRNA also inhibited the RelB DNA binding capacity but had no effect on other NF-κB subunits. The shRNA RelB-DCs can significantly inhibit mixed lymphocyte reaction (MLR) and down-regulate Th1 cytokines and prompt the production of Th2 cytokines. CONCLUSION: Our results indicate RelB shRNA transfection of DCs can induce the immature status, and produce tolerogenic DCs.