Antitumor Efficacy Of Natural DsRNA Combined With Panaxadiolsaponins (PDS) In Vitro And In Vivo

Prostate cancer is one of the most common malignance and the second leading cause of death among European and American men. Recently, with the ageing population, the changing life style and the development of detection, the incidence and mortality of prostate cancer in our country have bee increasing. Screening of prostate cancer groups in the male of fifty years old or over in ChangChun City, it was found that the case finding rate of prostate cancer is 1.7%, among which middle-advanced stage prostate cancer rate is 42%. So, prostate cancer has become the important menace for the health of old sterner sex in China. Hormone-refractory prostate cancer (HRPC) is one of difficulties in the clinic cure of prostate cancer, which has become the international problem. Therefore, the studies of promising antineoplastic drugs and biological therapies are very significant.IFN are not only antivirals with a spectrum of clinical effectiveness against viruses, but are also the prototypic biological responses modifies for oncology. Although the number of functional genes identified that encode type I IFNs (IFN-αand IFN-β) has grown significantly, this was only firmly established by the cloning of IFN-α1, IFN-α2 and IFN-β. Many researchers found that the cell factors can affect the immune activity of effector cell. The interferon (IFN) has been widely proved by inland and overseas study reports that it can inhibit virus diseases, malignant tumors and improve the immunoregulation ability effectively. IFN, as one of the important gene therapies, has been realized by many researchers recent year. IFN can directly affect tumor cell growth and apoptosis to restrain tumor growth by both controlling the immunity and regulating many biological factors. A great example from a random clinic control test show that IFN treatments after liver cancer operation can effectively decrease recurrence and improve live rate.Exogenous interferons can be induced in cells culture or through recombinant DNA production. However, development of therapeutic final products for human use has been hampered by the cost or limited by the few subtype production. For clinical utilization, dsRNA as IFN inducer has many advantages over the use of exogenous IFN. First, dsRNA possesses the potential for generating multiple species of IFNs. We consider there could be other biologic factors production induced directly or indirectly by dsRNA induction. Second, the sources of dsRNA are more widely presence than IFN production. Now the IFN inducers can be a wide variety of natural or synthetic agents, among which those most generally effective are viruses and certain high molecular weight, natural, or synthetic double-stranded RNAs. A Toll-like receptors (TLRs) function as pathogen pattern recognition molecules that sensor and initiate innate and adaptive immune responses against microbes and cancer cells. Recognition of pathogen-derived ligands by TLRs expressed on many types of cells, including dendritic cells and T cells, triggers the nuclear factor (NF)- B and type-1 interferon pathways, leading to the production of proinflammatory cytokines that are essential in stimulating CD4~+ T cells to differentiate to T helper (Th) 1, Th2 Th17 and regulatory T (Treg) cells.Toll-like receptor 3 (TLR3) recognizes viral dsRNA and its synthetic analog polyriboinosinc:polyribocytidylic acid (poly(I:C) and induces type I interferon (IFN), inflammatory cytokine/chemokine production and dentritic cell (DC) maturation. Natural long dsRNA is not only a kind of interferon inducer but also a broad-spectrum immunomodulator. It also has antivirus, anti-tumor and enhancing immunity functions. CTL and NK cells are principal effector cells in anti-viral and anti-tumor immunity. DsRNA, when added or transfected to cells, can rapidly induce transcription of many genes, including IFN genes. It can also downregulate the expression of another set of genes. The dsRNA-stimulated genes include IFN-stimulated genes and genes involved in TNF-induced signaling and apoptosis. They also include genes for cytokines and growth factors, RNA synthesis, protein sythesis and degradation, metabolism and biosynthesis, The dsRNA-repressed genes include genes involved in metabolism, cell cycle regulation, and cell adhesion. Long dsRNA can activate the IFN defense pathway and two IFN enzyme: one is 2',5'-OAS. The enhancing synthesis of 2',5'oligonucleotide can activate RNase and degrade mRNA because of the non-specificity of RNase. The other is long dsRNA-dependent PKR. In most mammalian somatic cells, PKR can be activated by dsRNA to autophosphorylate. Then eukaryotic initiation factor eIF2-αis phosphorylated, which leads to protein translation inhibition and cell apoptosis. Recently, PKR has attracted more attentions, which is seen as a new antitumor strategy.In this paper, long dsRNA extracted from yeast was combined with PDS to inhibit different tumor cells. The mechanism of the anti-tumor effects of dsRNA and PDS is discussed.Method1. Extraction and Identification of long dsRNA: The natural dsRNA was extracted from yeast fungus with hot phenol method and identified for its components. Using VSV virus inhibiting method and IFN ELISA kit we detected the effects of dsRNA-induced IFN.2. The inhibitory effects of dsRNA on mouse prostate cancer in vitro:2.1 MTT colorimetry was used to detect cell proliferation activity: RM-1 cells were divided into five groups as bellow: mock, dsRNA, PDS, dsRNA+PDS and Poly:IC as positive control. And the proliferation inhibition rates of every group are calculated.2.2 Apoptosis detection: After dsRNA and PDS treatments, using the flow cytometry we analyzed the cell cycle and apoptotic peak of RM-1; Using acridine orang stain we detected whether the jacinth fluorimetric apoptotic cells exist. Using tunnel fluorescent labeling we detected green fluorescence apoptotic cells.2.3 Western blot was used to detect the expressions of corresponding protein. We extracted cell proteins from every group (being the same as MTT groups), applied 10% SDS-PAGE electrophoresis, transferred to PVDF film, hybridizated and colorated with phosphorylated PKR, eIF2α, caspase 3, P53, VEGF polyclonal antibody.2.4 RT-PCR was used to detect the expression of corresponding genes. The whole cell RNA was extracted from each group (being the same as MTT groups), and used to amplify PKR, 2'5AOS, IFN, P21, bax, bcl2 genes with RT-PCR.3. The inhibitory effects of dsRNA on mouse prostate cancer in vivo:3.1 Preparation of RM-1 mouse bearing prostate cancer as model3.2 Tumor-bearing mice were divided into 5 groups: mock group, dsRNA group, PDS group, dsRNA+PDS group and PolyI:C group. PolyI:C group was used as the positive control. The biological changes of tumor were observed.3.3 Western blot and RT-PCR techniques were used to detected the expressions of corresponding gene and protein (The methods were same as the experiment in vitro).3.4 Using the immunohistochemistry we detected the expression of phosphorylated PKR, eIF2α, caspase 3, P53, VEGF expression. Using TUNEL kits detect apoptosis of tumor cells.4. In order to identify the anti-tumor effects of dsRNA, the dsRNA inhibitory effects on mouse melanoma B16 were detected in vitro and vivo (The methods were same as the above experiments).Results:1. The natural dsRNA was successfully extracted from yeast fungus and identified by electrophoresis and complete detects. And the IFN induction of dsRNA was detected by VSV virus inhibiting method and IFN ELISA kit.2. dsRNA significantly induced apoptosis effects on prostate cancer cells. MTT results showed that dsRNA combined with PDS showed synergistic antitumor cell growth effect. The apoptosis rate reaching to almost 39.9%, was found before the diploid cell G0/G1 peak by using flow cytometry analysis. Using acridine orange stain, there exist the jacinth fluorescent apoptotic cells. Green fluorescent apoptotic cells were also found by TUNEL kit detection. All these results indicated that dsRNA and PDS treatment can induce tumor cell apoptosis, which showed a significant difference compared with the control group (P<0.05). The semi-quantative RT-PCR results showed that dsRNA and PDS can enhance the expression of PKR, 2'5AOS, IFN, P21 and down-regulate the expression of bcl2 mRNA. Western blot results indicated the expression of phosphorylated PKR and eIF2αincreased significantly affected by dsRNA and PDS treatment compared with mock group. Meanwhile, the expressions of caspase 3 and P53 increased. The expressions ofβ-actin in every groups are not different obviously (P>0.05).3. RM-1 mouse bearing prostate cancer as a model was constructed. After treated with dsRNA and PDS, the volume and weight of tumor decreased obviously, which showed significantly difference compared with mock group (p<0.05). RT-PCR results indicated that dsRNA can enhance the expression of PKR, 2'5AOS, IFN, P21, caspase 3 and down-regulated the expression of bcl2 mRNA. Western blot results indicated the expressions of phosphorylated PKR and eIF2αincreased significantly affected by dsRNA and PDS, the expressions of caspase 3 and P53 were enhanced, while VEGF expression was down-regulated. Using TUNEL kits we detected apoptosis of tumor cells after treating with dsRNA and PDS. More importantly, dsRNA and PDS exhibit synergistic suppression effects on tumor cell growth significantly.4. The results of dsRNA inhibition experiments of mouse melanoma B16 and subcutaneously transplanting tumor experiments showed that dsRNA combined with PDS exhibited synergistic effects on inhibiting tumor cell growth and inducing apoptosis. In vitro and vivo experiments, the anti-tumor effects of dsRNA and PDS are similar: cell proliferation inhibitory rate and cell apoptosis rate are significantly higher than mock groups; the average weight and volume of tumors decrease in all cases. The results of semi-quantative RT-PCR and Western blot were similar as those shown in section 3.Conclusion: (1) The natural dsRNA extracted from yeast are excellent inducer of IFN; (2) dsRNA utilization showed a strong capacity for inhibition of tumor cell proliferation and inducing apoptosis effects on tumor cells; (3) The antitumor function of dsRNA are possibly related to the activation of PKR and phosphorylation of eIF2-αfollowing by the protein synthesis suppression and apoptosis. Meanwhile, dsRNA can enhance the expression of caspase 3, P21, P53 and bax, and inhibit the expression of anti- apoptosis proteins, such as bcl2 and VEGF, etc.In summary, the natural dsRNA can inhibit prostate cancer and melanoma cell growth. The anti-tumor effects are more significant when combining with PDS. This paper adopted first time to show the long natural dsRNA being extracted from yeast can induce tumor cell apoptosis. Currently, there are no internal and overseas analogous reports.