Enhancement of the Antitumor and Antimetastatic Effect of Topotecan and Normalization of Blood Counts in Mice with Lewis Carcinoma by Tdp1 Inhibitors-New Usnic Acid Derivatives.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an important DNA repair enzyme and one of the causes of tumor resistance to topoisomerase 1 inhibitors such as topotecan. Inhibitors of this Tdp1 in combination with topotecan may improve the effectiveness of therapy. In this work, we synthesized usnic acid derivatives, which are hybrids of its known derivatives: tumor sensitizers to topotecan. New compounds inhibit Tdp1 in the micromolar and submicromolar concentration range; some of them enhance the effect of topotecan on the metabolic activity of cells of various lines according to the MTT test. One of the new compounds (compound 7) not only sensitizes Krebs-2 and Lewis carcinomas of mice to the action of topotecan, but also normalizes the state of the peripheral blood of mice, which is disturbed in the presence of a tumor. Thus, the synthesized substances may be the prototype of a new class of additional therapy for cancer.

Comparing the Biological Properties of Double-Stranded DNA Extracted from Human and Porcine Placenta and Salmon Sperm.

Background: Double-stranded fragmented extracellular DNA is a participant, inducer, and indicator of various processes occurring in the organism. When investigating the properties of extracellular DNA, the question regarding the specificity of exposure to DNA from different sources has always been raised. The aim of this study was to perform comparative assessment of biological properties of double-stranded DNA obtained from the human placenta, porcine placenta and salmon sperm. Methods: The intensity of leukocyte-stimulating effect of different dsDNA was assessed in mice after cyclophosphamide-induced cytoreduction. The stimulatory effect of different dsDNA on maturation and functions of human dendritic cells and the intensity of cytokine production by human whole blood cells was analyzed ex vivo. The oxidation level of the dsDNA was also compared. Results: Human placental DNA exhibited the strongest leukocyte-stimulating effect. DNA extracted from human and porcine placenta exhibited similar stimulatory action on maturation of dendritic cells, allostimulatory capacity, and ability of dendritic cells to induce generation of cytotoxic CD8+CD107a+ T cells in the mixed leukocyte reaction. DNA extracted from salmon sperm stimulated the maturation of dendritic cells, while having no effect on their allostimulatory capacity. DNA extracted from human and porcine placenta was shown to exhibit a stimulatory effect on cytokine secretion by human whole blood cells. The observed differences between the DNA preparations can be caused by the total methylation level and are not related to differences in oxidation level of DNA molecules. Conclusions: Human placental DNA exhibited the maximum combination of all biological effects.

Impact of Double-Stranded RNA Internalization on Hematopoietic Progenitors and Krebs-2 Cells and Mechanism.

It is well-established that double-stranded RNA (dsRNA) exhibits noticeable radioprotective and radiotherapeutic effects. The experiments conducted in this study directly demonstrated that dsRNA was delivered into the cell in its native form and that it induced hematopoietic progenitor proliferation. The 68 bp synthetic dsRNA labeled with 6-carboxyfluorescein (FAM) was internalized into mouse hematopoietic progenitors, c-Kit+ (a marker of long-term hematopoietic stem cells) cells and CD34+ (a marker of short-term hematopoietic stem cells and multipotent progenitors) cells. Treating bone marrow cells with dsRNA stimulated the growth of colonies, mainly cells of the granulocyte-macrophage lineage. A total of 0.8% of Krebs-2 cells internalized FAM-dsRNA and were simultaneously CD34+ cells. dsRNA in its native state was delivered into the cell, where it was present without any signs of processing. dsRNA binding to a cell was independent of cell charge. dsRNA internalization was related to the receptor-mediated process that requires energy from ATP. Synthetic dsRNA did not degrade in the bloodstream for at least 2 h. Hematopoietic precursors that had captured dsRNA reinfused into the bloodstream and populated the bone marrow and spleen. This study, for the first time, directly proved that synthetic dsRNA is internalized into a eukaryotic cell via a natural mechanism.

The Lipophilic Purine Nucleoside-Tdp1 Inhibitor-Enhances DNA Damage Induced by Topotecan In Vitro and Potentiates the Antitumor Effect of Topotecan In Vivo.

The use of cancer chemotherapy sensitizers is a promising approach to induce the effect of clinically used anticancer treatments. One of the interesting targets is Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), a DNA-repair enzyme, that may prevent the action of clinical Topoisomerase 1 (Top1) inhibitors, such as topotecan (Tpc). Tdp1 eliminates covalent Top1-DNA (Top1c) complexes that appear under the action of topotecan and determines the cytotoxic effect of this drug. We hypothesize that Tdp1 inhibition would sensitize cells towards the effect of Tpc. Herein, we report the synthesis and study of lipophilic derivatives of purine nucleosides that efficiently suppress Tdp1 activity, with IC50 values in the 0.3-22.0 µM range. We also showed that this compound class can enhance DNA damage induced by topotecan in vitro by Comet assay on human cell lines HeLa and potentiate the antitumor effect of topotecan in vivo on a mice ascitic Krebs-2 carcinoma model. Thereby, this type of compound may be useful to develop drugs, that sensitize the effect of topotecan and reduce the required dose and, as a result, side effects.

Experimental Comparison of the In Vivo Efficacy of Two Novel Anticancer Therapies.

BACKGROUND/AIM: We compared the therapeutic efficacy of two recently developed experimental anticancer technologies: 1) in situ vaccination based on local immunotherapy with CpG oligonucleotides and anti-OX40 antibodies to activate antitumor immune response and 2) "Karanahan" technology [from the Sanskrit karana ('source') + han ('to kill')] based on the combined injection of cyclophosphamide and double-stranded DNA to eradicate cancer stem cells. MATERIALS AND METHODS: The anticancer approaches were compared on three types of mouse malignant tumors with different grades of immunogenicity: weakly immunogenic carcinoma Krebs-2, moderately immunogenic Lewis carcinoma, and highly immunogenic A20 В-cellular lymphoma. RESULTS: Our results indicated that in situ vaccination was the most effective against the highly immunogenic tumor А20. In addition, "Karanahan" demonstrated high efficiency in all types of tumors, regardless of their immunogenicity or size. CONCLUSION: "Karanahan" therapy showed higher efficacy relative to in situ vaccination with CpG oligonucleotides and anti-OX40 antibodies.

Characterization of biological peculiarities of the radioprotective activity of double-stranded RNA isolated from Saccharomyces сerevisiae.

THE PURPOSE OF THE ARTICLE: Protection from ionizing radiation is the most important component in the curing malignant neoplasms, servicing atomic reactors, and resolving the situations associated with uncontrolled radioactive pollutions. In this regard, discovering new effective radioprotectors as well as novel principles of protecting living organisms from high-dose radiation is the most important factor, determining the new approaches in medical and technical usage of radiation. MATERIALS AND METHODS: Experimental animals were irradiated on the γ-emitter (Cs137) with a dose of 9.4 Gy. Radioprotective properties of several agents (total RNA, single-stranded RNA, double-stranded RNA and B-190) were estimated by the survival/death rates of experimental animals within 30-90 d. Pathomorphological examination of internal organs end electron microscope assay was done on days 9-12 after irradiation. Cloning and other molecular procedures were performed accordingly to commonly accepted protocols. For assessment of the internalization of labeled nucleic acid, bone marrow cells were incubated with double-stranded RNA labeled with 6-FAM fluorescent dye. Cells with internalized double-stranded RNA were assayed using Axio Imager M1 microscope. In the other experiment, bone marrow cells after incubation with double-stranded RNA were stained with Cy5-labeled anti-CD34 antibodies and assayed using Axioskop 2 microscope. RESULTS: In this study, several biological features of the radioprotective action of double-stranded RNA are characterized. It was shown that 160 µg of the double-stranded RNA per mouse protect experimental animals from the absolutely lethal dose of γ-radiation of 9.4 Gy. In different experiments, 80-100% of irradiated animals survive and live until their natural death. Radioprotective properties of double-stranded RNA were found to be independent on its sequence, but strictly dependent on its double-stranded form. Moreover, double-stranded RNA must have 'open' ends of the molecule to exert its radioprotective activity. CONCLUSIONS: Experiments indicate that radioprotective effect of double-stranded RNA is tightly bound to its internalization into hematopoietic stem cells, which further repopulate the spleen parenchyma of irradiated mice. Actively proliferating progenitors form the splenic colonies, which further serve as the basis for restoration of hematopoiesis and immune function and determine the survival of animals received the lethal dose of radiation.

Promising New Inhibitors of Tyrosyl-DNA Phosphodiesterase I (Tdp 1) Combining 4-Arylcoumarin and Monoterpenoid Moieties as Components of Complex Antitumor Therapy.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an important DNA repair enzyme in humans, and a current and promising inhibition target for the development of new chemosensitizing agents due to its ability to remove DNA damage caused by topoisomerase 1 (Top1) poisons such as topotecan and irinotecan. Herein, we report our work on the synthesis and characterization of new Tdp1 inhibitors that combine the arylcoumarin (neoflavonoid) and monoterpenoid moieties. Our results showed that they are potent Tdp1 inhibitors with IC50 values in the submicromolar range. In vivo experiments with mice revealed that compound 3ba (IC50 0.62 µM) induced a significant increase in the antitumor effect of topotecan on the Krebs-2 ascites tumor model. Our results further strengthen the argument that Tdp1 is a druggable target with the potential to be developed into a clinically-potent adjunct therapy in conjunction with Top1 poisons.

Efficacy of a new cancer treatment strategy based on eradication of tumor-initiating stem cells in a mouse model of Krebs-2 solid adenocarcinoma.

Krebs-2 solid carcinoma was cured using a new "3+1" strategy for eradication of Krebs-2 tumor-initiating stem cells. This strategy was based on synchronization of these cells in a treatment-sensitive phase of the cell cycle. The synchronization mechanism, subsequent destruction of Krebs-2 tumor-initiating stem cells, and cure of mice from a solid graft were found to depend on the temporal profile of the interstrand cross-link repair cycle. Also, the temporal profile of the Krebs-2 interstrand repair cycle was found to have a pronounced seasonal cyclicity at the place of experiments (Novosibirsk, Russia). As a result, the therapeutic effect that is based on application of the described strategy, originally developed for the "winter repair cycle" (November-April), is completely eliminated in the summer period (June-September). We conclude that оne of the possible and the likeliest reasons for our failure to observe the therapeutic effects was the seasonal cyclicity in the duration of the interstrand repair cycle, the parameter that is central to our strategy.

Gene expression profiling of tumor-initiating stem cells from mouse Krebs-2 carcinoma using a novel marker of poorly differentiated cells.

Using the ability of poorly differentiated cells to natively internalize fragments of extracellular double-stranded DNA as a marker, we isolated a tumorigenic subpopulation present in Krebs-2 ascites that demonstrated the features of tumor-inducing cancer stem cells. Having combined TAMRA-labeled DNA probe and the power of RNA-seq technology, we identified a set of 168 genes specifically expressed in TAMRA-positive cells (tumor-initiating stem cells), these genes remaining silent in TAMRA-negative cancer cells. TAMRA+ cells displayed gene expression signatures characteristic of both stem cells and cancer cells. The observed expression differences between TAMRA+ and TAMRA- cells were validated by Real Time PCR. The results obtained corroborated the biological data that TAMRA+ murine Krebs-2 tumor cells are tumor-initiating stem cells. The approach developed can be applied to profile any poorly differentiated cell types that are capable of immanent internalization of double-stranded DNA.

Five-year disease-free survival among stage II-IV breast cancer patients receiving FAC and AC chemotherapy in phase II clinical trials of Panagen.

BACKGROUND: We report on the results of a phase II clinical trial of Panagen (tablet form of fragmented human DNA preparation) in breast cancer patients (placebo group n = 23, Panagen n = 57). Panagen was administered as an adjuvant leukoprotective agent in FAC and AC chemotherapy regimens. Pre-clinical studies clearly indicate that Panagen acts by activating dendritic cells and induces the development of adaptive anticancer immune response. METHODS: We analyzed 5-year disease-free survival of patients recruited into the trial. RESULTS: Five-year disease-free survival in the placebo group was 40 % (n = 15), compared with the Panagen arm - 53 % (n = 51). Among stage III patients, disease-free survival was 25 and 52 % for placebo (n = 8) and Panagen (n = 25) groups, respectively. Disease-free survival of patients with IIIB + C stage was as follows: placebo (n = 6)-17 % vs Panagen (n = 18)-50 %. CONCLUSIONS: Disease-free survival rate (17 %) of patients with IIIB + C stage breast cancer receiving standard of care therapy is within the global range. Patients who additionally received Panagen demonstrate a significantly improved disease-free survival rate of 50 %. This confirms anticancer activity of Panagen. TRIAL REGISTRATION: ClinicalTrials.gov NCT02115984 from 04/07/2014.

Nonadherent Spheres With Multiple Myeloma Surface Markers Contain Cells that Contribute to Sphere Formation and Are Capable of Internalizing Extracellular Double-Stranded DNA.

BACKGROUND: The most prominent features of cancer stem cells are asymmetric cell division, tumorigenicity, and clonogenicity. Recently one more feature of poorly differentiated cell types of various origin, including cancer stem cells, has been described. Namely, these cells can internalize extracellular DNA natively, without additional transfection procedures. PATIENTS AND METHODS: Using our approach to trace internalization of a TAMRA (carboxy tetramethyl-rhodamine [fluorescent dye])-DNA labeled probe by poorly differentiated cell types, we isolated and characterized the cells from free-floating spheres derived from the bone marrow clonogenic aspirate of a multiple myeloma patient. RESULTS: Nonadherent spheres display a B-cell phenotype (CD73/CD20+/CD45+/CD19dim). Further, free-floating spheres contain 1% to 3% cells with a clonogenic potential, and these cells display a marker of poorly differentiated cell types (TAMRA+). Upon association with a group of ∼ 10 free-floating TAMRA- cells, this peculiar cell type forms a sphere-forming cluster that initiates secondary aggregation of cells into a spheric structure. TAMRA+ and TAMRA- cells secrete distinct sets of cytokines indicative of the paracrine regulation. Grafting experiments of intact whole spheres versus cell suspensions prepared from dispersed spheres indicate that successful engraftment only occurs in the former case. CONCLUSION: Nonadherent 3-D cell colonies (spheres) encompass B cells with CD73/CD20+/CD45+/CD19dim phenotype, as well as double-stranded DNA-internalizing cells. The latter cell type appears to function as a sphere-forming center. Different cells in the spheres communicate with each other by secreting specific sets of cytokines. For successful engraftment and tumor growth in mice, intact spheres containing ∼ 106 cells must be used.

Properties of internalization factors contributing to the uptake of extracellular DNA into tumor-initiating stem cells of mouse Krebs-2 cell line.

BACKGROUND: Previously, we demonstrated that poorly differentiated cells of various origins, including tumor-initiating stem cells present in the ascites form of mouse cancer cell line Krebs-2, are capable of naturally internalizing both linear double-stranded DNA and circular plasmid DNA. METHODS: The method of co-incubating Krebs-2 cells with extracellular plasmid DNA (pUC19) or TAMRA-5'-dUTP-labeled polymerase chain reaction (PCR) product was used. It was found that internalized plasmid DNA isolated from Krebs-2 can be transformed into competent Escherichia coli cells. Thus, the internalization processes taking place in the Krebs-2 cell subpopulation have been analyzed and compared, as assayed by E. coli colony formation assay (plasmid DNA) and cytofluorescence (TAMRA-DNA). RESULTS: We showed that extracellular DNA both in the form of plasmid DNA and a PCR product is internalized by the same subpopulation of Krebs-2 cells. We found that the saturation threshold for Krebs-2 ascites cells is 0.5 µg DNA/10(6) cells. Supercoiled plasmid DNA, human high-molecular weight DNA, and 500 bp PCR fragments are internalized into the Krebs-2 tumor-initiating stem cells via distinct, non-competing internalization pathways. Under our experimental conditions, each cell may harbor 340-2600 copies of intact plasmid material, or up to 3.097 ± 0.044×10(6) plasmid copies (intact or not), as detected by quantitative PCR. CONCLUSION: The internalization dynamics of extracellular DNA, copy number of the plasmids taken up by the cells, and competition between different types of double-stranded DNA upon internalization into tumor-initiating stem cells of mouse ascites Krebs-2 have been comprehensively analyzed. Investigation of the extracellular DNA internalization into tumor-initiating stem cells is an important part of understanding their properties and possible destruction mechanisms. For example, a TAMRA-labeled DNA probe may serve as an instrument to develop a target for the therapy of cancer, aiming at elimination of tumor stem cells, as well as developing a straightforward test system for the quantification of poorly differentiated cells, including tumor-initiating stem cells, in the bulk tumor sample (biopsy or surgery specimen).

Antitumor and Antimetastatic Effect of Small Immunostimulatory RNA against B16 Melanoma in Mice.

Small interfering RNAs, depending on their structure, delivery system and sequence, can stimulate innate and adaptive immunity. The aim of this study was to investigate the antitumor and antimetastatic effects of immunostimulatory 19-bp dsRNA with 3'- trinucleotide overhangs (isRNA) on melanoma B16 in C57Bl/6 mice. Recently developed novel cationic liposomes 2X3-DOPE were used for the in vivo delivery of isRNA. Administration of isRNA/2X3-DOPE complexes significantly inhibits melanoma tumor growth and metastasis. Histopathological analysis of spleen cross sections showed hyperplasia of the lymphoid white pulp and formation of large germinal centers after isRNA/2X3-DOPE administration, indicating activation of the immune system. The treatment of melanoma-bearing mice with isRNA/2X3-DOPE decreases the destructive changes in the liver parenchyma. Thus, the developed isRNA displays pronounced immunostimulatory, antitumor and antimetastatic properties against melanoma B16 and may be considered a potential agent in the immunotherapy of melanoma.

A strategy to eradicate well-developed Krebs-2 ascites in mice.

We describe the strategy, which allows curing experimental mice engrafted with Krebs-2 ascites. The strategy is based on the facts that i) Krebs-2 tumor-initiating stem cells (TISCs) are naturally capable of internalizing fragments of extracellular double-stranded DNA (dsDNA); ii) upon delivery into TISCs, these dsDNA fragments interfere with the on-going DNA repair process so that TISCs either die or lose their tumorigenic potential. The following 3-step regimen of therapeutic procedures leading to eradication of Krebs-2 ascites is considered. Firstly, three timed injections of cyclophosphamide (CP) exactly matching the interstrand cross-link (ICL) repair phases that lead to synchronization of ascites cells in late S/G2/M. Secondly, additional treatment of ascites 18 hours post each CP injection (at NER/HR transition timepoint) with a composite dsDNA-based preparation interfering with the NER and HR repair pathways, so that tumorigenic properties of ascites cells are compromised. Thirdly, final treatment of mice with a combination of CP and dsDNA injections as ascites cells undergo apoptotic destruction, and the surviving TAMRA+ TISCs arrested in late S/G2/M phases massively enter into G1/S, when they regain sensitivity to CP+dsDNA treatment. Thus, this regimen assures that no viable cells, particularly Krebs-2 TISCs, remain.

Comparative analysis of pathologic processes developing in mice housed in SPF vs non-SPF conditions and treated with cyclophosphamide and dsDNA preparation.

In our earlier studies, we observed that when mice are treated with cyclophosphamide and fragmented exogenous dsDNA (18-30 h post cytostatic treatment), they develop a very characteristic set of symptoms and 80-90% of such animals succumb within 6-25 days. This was called "delayed death" phenomenon, and the gap between cyclophosphamide and DNA injections required for such phenotype to develop was termed "death window". We established that mice succumbed to multi-organ failure, which was caused by systemic inflammation and sepsis. These processes unfolded along with accidental involution of lymphoid organs, which resulted from the failure of CD34(+) hematopoietic stem cells to differentiate into lymphoid lineage progenitors. Here we compare SPF and non-SPF animals, and demonstrate that the major cause of systemic inflammation and sepsis observed upon such treatments is activation of an opportunistic infection. Mice of the same strain (CBA) housed under SPF conditions do not develop the characteristic symptoms, nor do they become moribund. Yet, regardless of the breeding conditions, upon synergistic action of cyclophosphamide and dsDNA, CD34(+) hematopoietic stem cells consistently fail to give rise to lymphoid lineage progenitors. We demonstrate that this differentiation defect is reversible and that population of lymphoid progenitors is restored by day 29 after cyclophosphamide injection.

Results of multicenter double-blind placebo-controlled phase II clinical trial of Panagen preparation to evaluate its leukostimulatory activity and formation of the adaptive immune response in patients with stage II-IV breast cancer.

BACKGROUND: We performed a multicenter, double-blind, placebo-controlled, phase II clinical trial of human dsDNA-based preparation Panagen in a tablet form. In total, 80 female patients with stage II-IV breast cancer were recruited. METHODS: Patients received three consecutive FAC (5-fluorouracil, doxorubicin and cyclophosphamide) or AC (doxorubicin and cyclophosphamide) adjuvant chemotherapies (3 weeks per course) and 6 tablets of 5 mg Panagen or placebo daily (one tablet every 2-3 hours, 30 mg/day) for 18 days during each chemotherapy course. Statistical analysis was performed using Statistica 6.0 software, and non-parametric analyses, namely Wilcoxon-Mann-Whitney and paired Wilcoxon tests. To describe the results, the following parameters were used: number of observations (n), median, interquartile range, and minimum-maximum range. RESULTS: Panagen displayed pronounced leukostimulatory and leukoprotective effects when combined with chemotherapy. In an ancillary protocol, anticancer effects of a tablet form of Panagen were analyzed. We show that Panagen helps maintain the pre-therapeutic activity level of innate antitumor immunity and induces formation of a peripheral pool of cytotoxic CD8+ perforin + T-cells. Our 3-year follow-up analysis demonstrates that 24% of patients who received Panagen relapsed or died after the therapy, as compared to 45% in the placebo cohort. CONCLUSIONS: The data collected in this trial set Panagen as a multi-faceted "all-in-one" medicine that is capable of simultaneously sustaining hematopoiesis, sparing the innate immune cells from adverse effects of three consecutive rounds of chemotherapy and boosting individual adaptive immunity. Its unique feature is that it is delivered via gastrointestinal tract and acts through the lymphoid system of intestinal mucosa. Taken together, maintenance of the initial levels of innate immunity, development of adaptive cytotoxic immune response and significantly reduced incidence of relapses 3 years after the therapy argue for the anticancer activity of Panagen. TRIAL REGISTRATION: ClinicalTrials.gov NCT02115984 from 04/07/2014.

Combination of cyclophosphamide and double-stranded DNA demonstrates synergistic toxicity against established xenografts.

BACKGROUND: Extracellular double-stranded DNA participates in various processes in an organism. Here we report the suppressive effects of fragmented human double-stranded DNA along or in combination with cyclophosphamide on solid and ascites grafts of mouse Krebs-2 tumor cells and DNA preparation on human breast adenocarcinoma cell line MCF-7. METHODS: Apoptosis and necrosis were assayed by electrophoretic analysis (DNA nucleosomal fragmentation) and by measurements of LDH levels in ascitic fluid, respectively. DNA internalization into MCF-7 was analyzed by flow cytometry and fluorescence microscopy. RESULTS: Direct cytotoxic activity of double-stranded DNA (along or in combination with cyclophosphamide) on a solid transplant was demonstrated. This resulted in delayed solid tumor proliferation and partial tumor lysis due to necrosis of the tumor and adjacent tissues. In the case of ascites form of tumor, extensive apoptosis and secondary necrosis were observed. Similarly, MCF-7 cells showed induction of massive apoptosis (up to 45%) as a result of treatments with double-stranded DNA preparation. CONCLUSIONS: Double-stranded DNA (along or in combination with cyclophosphamide) induces massive apoptosis of Krebs-2 ascite cells and MCF-7 cell line (DNA only). In treated mice it reduces the integrity of gut wall cells and contributes to the development of systemic inflammatory reaction.

Identification of cancer stem cells and a strategy for their elimination.

It has been established previously that up to 40% of mouse CD34(+) hematopoietic stem cells are capable of internalizing exogenous dsDNA fragments both in vivo and ex vivo. Importantly, when mice are treated with a combination of cyclophosphamide and dsDNA, the repair of interstrand crosslinks in hematopoietic progenitors is attenuated, and their pluripotency is altered. Here we show for the first time that among various actively proliferating mammalian cell populations there are subpopulations capable of internalizing dsDNA fragments. In the context of cancer, such dsDNA-internalizing cell subpopulations display cancer stem cell-like phenotype. Furthermore, using Krebs-2 ascites cells as a model, we found that upon combined treatment with cyclophosphamide and dsDNA, engrafted material loses its tumor-initiating properties which we attribute to the elimination of tumor-initiating stem cell subpopulation or loss of its tumorigenic potential.

Immunotherapy of hepatocellular carcinoma with small double-stranded RNA.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with limited therapeutic options. Since HCC has been shown to be immunogenic, immunotherapy is considered a promising therapeutic approach. Small interfering RNAs (siRNAs), depending on their structure and sequence, can trigger the innate immune system, which can potentially enhance the adaptive anticancer immune response in the tumor-bearing subjects. Immunostimulatory properties of nucleic acids can be applied to develop adjuvants for HCC treatment. METHODS: The transplantable HCC G-29 tumor in male CBA/LacSto (CBA) mice was used to study the effects of immunostimulatory RNA on tumor growth. Tumor size, metastases area in different organs of mice and mouse survival rate were analyzed. Furthermore the mouse serum IFN-α levels were measured using ELISA. RESULTS: In the present study, we found that a 19-bp RNA duplex (ImmunoStimulattory RNA or isRNA) with 3-nt overhangs at the 3'-ends of specific sequence displays immunostimulatory, antitumor, and antimetastatic activities in mice bearing HCC G-29. Our results demonstrate that isRNA strongly increases the level of interferon-α (IFN-α) by up to 25-fold relative to the level in mice injected with Lipofectamine alone (Mock), and to a lesser extent increases the level of proinflammatory cytokine interleukin-6 (IL-6) (by up to 5.5-fold relative to the Mock level), in mice blood serum. We showed that isRNA reliably (P < 0.05) inhibits primary tumor growth in mice compared to the mock group. Furthermore, injections of isRNA significantly enhanced necrotic processes in the center of the primary tumor, and decreased by twofold the width of the undifferentiated peripheral zone and the number of mitotic cells in this zone. The results showed that isRNA efficiently reduces the area of metastases in the liver, kidneys, and heart of CBA/LacSto mice with HCC. CONCLUSIONS: The obtained results clearly demonstrate immunostimulatory and antimetastatic properties of the isRNAs in mice with HCC. Consequently, this short double-stranded RNA can be considered as a potential adjuvant for the therapy of HCC.