Synergistic inhibitory effect of resveratrol and TK/GCV therapy on melanoma cells.

PURPOSE: To investigate the synergistic effect of resveratrol on the bystander effect of TK/GCV suicide gene system in melanoma cells. METHODS: The effect of resveratrol on the growth of B16 cells and the synergistic effect of resveratrol with or without GCV were detected by MTT assay and high content screening assay. The effect of resveratrol on GJIC function was detected by flow cytometry combined with fluorescence tracer and fluorescence microscope, and the expression of gap junction protein was detected by western blotting. Synergistic killing effect of resveratrol plus TK/GCV was tested in vivo using transplanted melanoma model. RESULTS: In vitro, resveratrol can enhanced GJ function and upregulated Cx32 and Cx43 protein expression in B16 cells. Resveratrol synergized with GCV to kill mixed B16 melanoma cells (20% TK+ cells and 80% TK- cells) and to improve apoptosis rate of TK- cells (the bystander effect of TK system), and the synergistic action was reversed by the GJ inhibitor AGA. In vivo, when B16 cells were mixed with 30% TK+ B16 cells, significantly reduced tumor weight and volume were observed after combinational treatment with resveratrol plus GCV as compared with GCV or resveratrol treatment alone. CONCLUSIONS: Resveratrol could synergistically enhance the killing effect of TK/GCV suicide gene system in melanoma B16 cells and transplanted melanoma. It might be a promising adjuvant of TK/GCV therapy.

Curcumin plays a synergistic role in combination with HSV-TK/GCV in inhibiting growth of murine B16 melanoma cells and melanoma xenografts.

Melanoma is a global concern and accounts for the major mortality of skin cancers. Herpes simplex virus thymidine kinase gene with ganciclovir (HSV-TK/GCV) is a promising gene therapy for melanoma. Despite its low efficiency, it is well known for its bystander effect which is mainly mediated by gap junction. In this study, we found that curcumin reduced B16 melanoma cell viability in both time- and dose-dependent manner. Further study showed that curcumin improved the gap junction intercellular communication (GJIC) function, and upregulated the proteins essential to gap junction, such as connexin 32 and connexin 43, indicating the potential role in enhancing the bystander effect of HSV-TK/GCV. By co-culturing the B16TK cells, which stably expressed TK gene, with wildtype B16 (B16WT) cells, we found that co-treatment of curcumin and GCV synergistically inhibited B16 cell proliferation, but the effect could be eliminated by the gap junction inhibitor AGA. Moreover, curcumin markedly increased apoptosis rate of B16WT cells, suggesting its effect in enhancing the bystander effect of HSV-TK/GCV. In the in-vivo study, we established the xenografted melanoma model in 14 days by injecting mixture of B16TK and B16WT cell in a ratio of 3:7. The result demonstrated that, co-administration of curcumin and GCV significantly inhibited the xenograft growth, as indicated by the smaller size and less weight. The combinational effect was further confirmed as a synergistic effect. In conclusion, the results demonstrated that curcumin could enhance the killing effect and the bystander effect of HSV-TK/GCV in treating melanoma, which might be mediated by improved gap junction. Our data suggested that combination of HSV-TK/GCV with curcumin could be a potential chemosensitization strategy for cancer treatment.

Synergistic effect of resveratrol and HSV-TK/GCV therapy on murine hepatoma cells.

Despite its low transfer efficiency, suicide gene therapy with HSV-TK is known for its bystander killing effect. The connexin-based gap junction is believed to mediate the bystander effect. Recently, we found that resveratrol, a polyphenol compound, increased the expression of Cx26 and Cx43, which are connexins and important constituents of gap junctions, in murine hepatoma cells. Hypothetically, the resveratrol-induced upregulation of gap junctions may improve the bystander effect that HSV-TK/GCV has on hepatoma cells. Our present investigation revealed that resveratrol could enhance intercellular communication at the gap junctions in CBRH7919 hepatoma cells and thereby enhance the bystander killing effect of GCV on CBRH7919TK cells. However, inhibition of gap junction using its long-term inhibitor alpha-glycyrrhetinic acid had a negative influence on the bystander effect of gene therapy with HSV-TK/GCV. In addition, combined resveratrol and GCV treatment in tumor-bearing mice with CBRH7919TK and CBRH7919WT cells at a ratio of 2:3 resulted in a significant decrease in the volume and weight of the tumor in comparison to GCV or only resveratrol. The present results demonstrate that resveratrol can enhance the bystander effect exerted by the HSV-TK/GCV system by enhancing connexin-mediated gap junctional communication.

A Circular RNA Binds To and Activates AKT Phosphorylation and Nuclear Localization Reducing Apoptosis and Enhancing Cardiac Repair.

As central nodes in cardiomyocyte signaling, nuclear AKT appears to play a cardio-protective role in cardiovascular disease. Here we describe a circular RNA, circ-Amotl1 that is highly expressed in neonatal human cardiac tissue, and potentiates AKT-enhanced cardiomyocyte survival. We hypothesize that circ-Amotl1 binds to PDK1 and AKT1, leading to AKT1 phosphorylation and nuclear translocation. In primary cardiomyocytes, epithelial cells, and endothelial cells, we found that forced circ-Amotl1 expression increased the nuclear fraction of pAKT. We further detected increased nuclear pAKT in circ-Amotl1-treated hearts. In vivo, circ-Amotl1 expression was also found to be protective against Doxorubicin (Dox)-induced cardiomyopathy. Putative PDK1- and AKT1-binding sites were then identified in silico. Blocking oligonucleotides could reverse the effects of exogenous circ-Amotl1. We conclude that circ-Amotl1 physically binds to both PDK1 and AKT1, facilitating the cardio-protective nuclear translocation of pAKT.

Dioscin augments HSV-tk-mediated suicide gene therapy for melanoma by promoting connexin-based intercellular communication.

Suicide gene therapy is a promising strategy against melanoma. However, the low efficiency of the gene transfer technique can limit its application. Our preliminary data showed that dioscin, a glucoside saponin, could upregulate the expression of connexins Cx26 and Cx43, major components of gap junctions, in melanoma cells. We hypothesized that dioscin may increase the bystander effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) through increasing the formation of gap junctions. Further analysis showed that dioscin indeed could increase the gap junctional intercellular communication in B16 melanoma cells, resulting in more efficient GCV-induced bystander killing in B16tk cells. By contrast, overexpression of dominant negative Cx43 impaired the cell-cell communication of B16 cells and subsequently weakened the bystander effect of HSV-tk/GCV gene therapy. In vivo, combination treatment with dioscin and GCV of tumor-bearing mice with 30% positive B16tk cells and 70% wild-type B16 cells caused a significant reduction in tumor volume and weight compared to treatment with GCV or dioscin alone. Taken together, these results demonstrated that dioscin could augment the bystander effect of the HSV-tk/GCV system through increasing connexin-mediated gap junction coupling.

Dioscin suppresses hepatocellular carcinoma tumor growth by inducing apoptosis and regulation of TP53, BAX, BCL2 and cleaved CASP3.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most commonly diagnosed malignancy of the liver, occurs frequently in the setting of chronic liver injury. Although multiple therapeutic approaches are available, the prognosis of patients with HCC remains poor. Dioscin is a natural steroid saponin that presents in various plants. The anti-cancer and anti-fibrotic effects have been extensively reported. However, the effect of dioscin on HCC remains unclear. We aimed to investigate the anti-HCC properties of dioscin in vitro and in vivo. METHODS: MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide) assay was used to analyze the growth inhibition activity of Dioscin in human cell lines, Bel-7402, HepG2, Lovo, and EAhy926. Antitumor activity through induction of apoptosis was evaluated by flow cytometry using Annexin-V and propidium iodide (PI) staining, laser scanning confocal microscopy (LSCM) analysis with Hochest33342 and PI labeling, and DNA fragmentation analysis. The expression of apoptosis-related proteins tumor protein p53 (TP53), BCL2-associated X protein (BAX), B-Cell CLL/Lymphoma 2 (BCL2) and Caspase 3 (CASP3) was measured by Western blot. Nude mice bearing Bel-7402 were administered intraperitoneally at different doses of dioscin and 5-FU (5-Fluorouracil) treatment was used as a control. Tumor volume and tumor weight of each mouse were then measured. RESULTS: We demonstrated that Dioscin inhibited proliferation of HCC cell lines in a dose-dependent manner. Dioscin also significantly induced morphological changes during death by apoptosis and increased DNA damage of Bel-7402 cells. Moreover, we demonstrated that Dioscin displayed anticancer activity via up-regulating expression of TP53, BAX and CASP3 protein, as well as down-regulating BCL2 in Bel-7402 cells. Notably, the in vivo anticancer activity of Dioscin was further assessed and achieved greater inhibition efficiency at the concentration increased to 24mg/kg/day than 5-FU at dose of 10mg/kg/day in nude mice bearing Bel-7402 cells. CONCLUSIONS: Dioscin inhibited tumor growth via inducing apoptosis, which was accompanied by altered expression of apoptotic pathway proteins, such as TP53, BAX, BCL2 and CASP3. Our findings indicate that further evaluation of dioscin as a novel therapeutic approach for HCC is warranted.

[The preliminary research on the gap junction mechanisms for synergistic effects of liuwei di-huang pill containing serum on suicide gene therapy of melanoma].

OBJECTIVE: To study the gap junction mechanisms for synergistic effects of liuwei di-huang pill (LDP) containing serum on HSV-tk/GCV suicide gene therapy of mouse malignant melanoma B16 cells. METHODS: The LDP containing serum (2.5% LDP serum group, 5.0% LDP serum group, and 10.0% LDP serum group) and the control serum group were set up. The effects of LDP on mRNA expressions of Cx26 and Cx43 in mouse malignant melanoma B16 cells were detected by RT-PCR assay. The effects of LDP on protein expressions of Cx26 and Cx43 in B16 cells were detected using Western blot and indirect immunofluorescence assay. The interference efficiencies of Cx26-309, Cx26-337, Cx26-367, Cx26-2098 SiRNA on Cx26 gene in B16 cells were detected using RT-RCR technique. Cx26-2098 SiRNA, due to the optimal interference efficiency, was chosen to interfere Cx26 gene, and the bystander effect of LDP + HSV-tk/GCV was observed. The effects of the gap junctional intercellular communication (GJIC) inhibitor glycyrrhetinic acid on the killing of LDP + HSV-tk/GCV system to B16 cells were detected by MTT assay. In this experiment, the control serum group, 2.5% LDP serum group, 5. 0% LDP serum group, 10.0% LDP serum group, the control serum combined GCV group, 2.5% LDP serum combined GCV group, 5.0% LDP serum combined GCV group, 10.0% LDP serum combined GCV group, 2.5% LDP serum combined GCV + glycyrrhetinic acid group, 5.0% LDP serum combined GCV + glycyrrhetinic acid group, 10.0% LDP serum combined GCV + glycyrrhetinic acid group were set up. The final concentration of GCV was 20 micromol/L. The final concentration of glycyrrhetinic acid was 50 micromol/L. RESULTS: LDP containing serum could increase the protein and mRNA expressions of Cx43 in B16 cell in a dose-dependent manner. It had bi-directional regulation on the Cx26 protein and mRNA expressions. The low dose LDP had inhibition while high dose LDP could up-regulate its expression. After SiRNA interfered Cx26 gene, there was no obvious change in the bystander effect of LDP combined suicide gene therapy between before and after interference. There was significant reduction in the inhibition rate between before (48.75%, 59.39%, and 69.28%) and after blockage (29.14%, 38.71%, and 58.13%) of glycyrrhetinic acid in the 2. 5%, 5.0%, 10.0% LDP serum combined GCV groups (P <0.01). CONCLUSION: The synergistic effects of LDP containing serum on HSV-tk/GCV suicide gene therapy in mouse malignant melanoma B16 cells were correlated with the gap junction mechanisms, which might be achieved through increasing the expressions of Cx26 and Cx43.

Tanshinone IIA increases the bystander effect of herpes simplex virus thymidine kinase/ganciclovir gene therapy via enhanced gap junctional intercellular communication.

The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV). This effect is reported to be mediated by gap junctional intercellular communication (GJIC), and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA), a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy.

Induction of apoptosis by evodiamine involves both activation of mitotic arrest and mitotic slippage.

Evodiamine (Evo) is an indole quinazoline alkaloid isolated from the fruit of Evodia rutaecarpa Bentham. Previous studies have shown that Evo exhibits anti-proliferative anti-tumor activities in several cancer types, but its target(s) and underlying mechanism(s) of action remain unclear. In the present study, we sought to establish a cell synchronization model in order to examine the anti-proliferative and apoptotic mechanisms of Evo in the human gastric cancer cell line SGC-7901. In addition, we transfected these cells with full-length or non-degradable (ND) cyclinB1 to evaluate the relationship between the induction of apoptosis and activation of mitotic arrest and mitotic slippage by Evo. Our results demonstrated that Evo markedly inhibited cell growth and was cytotoxic to SGC-7901 cells. Furthermore, transient Evo treatment (<16 h) caused reversible mitotic arrest, but sustained mitotic arrest was required to initiate apoptosis. The time required to reverse the apoptotic effects of Evo was between 16 and 20 h. We also demonstrated that promotion of mitotic slippage by a CDK1 inhibitor enhanced apoptosis. Furthermore, we evaluated the effect of delaying mitotic slippage by overexpressing ND cyclinB1, which delayed apoptosis. In conclusion, these results indicate that Evo-induced apoptosis is associated with mitotic arrest and subsequent mitotic slippage, which may underlie the actions of Evo in the treatment and prevention of cancer.

[Preliminary evaluation of immunological function in patients with acute lymphocytic leukemia].

This study was aimed to investigate 6 kinds of human cytokines (IL-2, IL-4, IL-12, IL-13, IFN-gamma and TNF-alpha) in patients with acute lymphocytic leukemia (ALL), so as to find their relationship with the disease. The pure monoclonal antibodies were spotted on the prepared NC membrane glass slides under certain conditions to make human cytokine protein microarray. The serum samples were collected from peripheral blood of 28 patients and 25 normal controls, the serum concentration of cytokines was determined by using the protein microarray and ELISA. The results showed that the expression levels of IL-4, IL-12, IL-13, IFN-gamma and TNF-alpha in ALL patients were lower than those in the normal controls, but ELISA indicated that the expression of TNF-alpha in ALL patients and normal controls was negative. In conclusion, both cellular and humoral immunity are all inhibited in patients with ALL. Microarray is more sensitive than ELISA.