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Introduction. Circadian rhythms are an adaptational mechanism to day-night cycle. At the cellular level rhythms are supported by oscillations of transcription of Bmal1, 2, Clock and Npas2, Per1-3, Cry1, 2 clock genes; in the organism rhythms are synchronized with melatonin, the "hormone of darkness". Reliable data have been obtained that disruption of biological "clocks" at the cellular and/or organism level are associated with carcinogenesis, but the experimental results in this direction remain incomplete. Aim. Assessment of the continuous lighting and melatonin administration effects on chemically induced carcinogenesis in mice and expression of clock genes and proteins in normal and tumor lung tissue. Materials and methods. Lung tumors were induced by urethane in 120 male SHR mice. Animals were kept at constant (LL) or standard (LD) light; half of the animals received melatonin (MT) daily at 20 mg/l at night with water for 45 weeks of experiment. At the end of the experiment, the frequency and multiplicity of lung tumours of different size were assessed. Expression of clock genes Clock, Bmal1, Cry1 was assessed in tumor samples and normal lung tissue by real-time PCR; protein content of BMAL1, CLOCK, CRY1 and PER2 was determined by immunohistochemistry. Statistical data were processed using the programs MS Excel 2007, GraphPad Prism 6.0 with commonly used statistical criteria. Results. The number of animals with lung tumors was between 89 and 100% in different groups. Large tumors (>2 mm) were more frequently observed in LL group (62 tumors out of 294, 21,1%) than in LD group (59 tumors out of 405, 14,6%, p=0,0245). Administration of exogenous melatonin at constant lighting statistically significantly reduced the frequency of large tumors (28 tumors out of 320, 8.8%, p=0,0001 in comparison with LL). In normal lung tissue of animals kept at constant lighting (LL) the increase in relative expression of clock genes in comparison with LD group was revealed: Bmal1 by 3,1 times (p=0,02) and Cry1 by 3,6 times (p=0,0002). No such differences were found for Clock gene. The relative content of BMAL1 and CLOCK proteins in all experimental conditions was higher in adenomas and adenocarcinomas compared to normal tissue. Conclusion. Constant lighting promotes the development of chemically induced lung tumors, and melatonin administration inhibits carcinogenesis under constant lighting. The content of BMAL1 and CLOCK transcription activator proteins in lung tumors was found to increase in comparison with normal tissue, whereas no increase in the expression level of corresponding genes in tumors was observed.

Galina Kireeva,1 Ekaterina Gubareva,1 Mikhail Maydin,1 Vladislav Osetnik,2 Stepan Kruglov,1 Andrey Panchenko,1 Anastasia Dorofeeva,1 Margarita Tyndyk,1 Elena Fedoros,1 Vladimir Anisimov1 1Department of Carcinogenesis and Aging, N.N. Petrov National Medical Research Center of Oncology, Saint-Petersburg, Russia; 2Surgical Department, Saint-Petersburg State University Hospital, Saint-Petersburg, RussiaCorrespondence: Galina KireevaDepartment of Carcinogenesis and Aging, N.N. Petrov National Medical Research Center of Oncology, Leningradskaya Str. 68. Pesochny, Saint-Petersburg, 197758, RussiaEmail galinakireyeva@mail.ruAim: Alterations in circadian rhythms caused by tumor growth are thought to be clinically relevant as they affect the prognosis and treatment response. We aimed to evaluate the chronotherapeutic approach in rats with ovarian cancer receiving cisplatin intravenously (IV) or with hyperthermic intraperitoneal chemoperfusion (HIPEC) and to assess daily variations in tumor and intestinal epithelium proliferation.Methods: In the pilot study, we used 12 intact rats and 12 rats with transplantable ovarian cancer, which were euthanized at ZT0 (08:00, lights on), ZT6, ZT12 and ZT18. In the main study, we used 45 rats with transplantable ovarian cancer. Animals were randomized into five groups: control, HIPEC with cisplatin at ZT0 (08:00), HIPEC with cisplatin at ZT12 (20:00), IV cisplatin at ZT0 and IV cisplatin at ZT12. We assessed the proliferation rate of tumor and small intestinal epithelium, apoptosis in small intestinal epithelium, and levels of γ-H2AX (DNA damage/repair marker) in kidneys and liver. Survival was calculated in each group.Results: Ascitic ovarian cancer disrupted daily variations in intestinal epithelium proliferation and DNA damage/repair in rats. Ovarian carcinoma exhibited no daily variation in mitotic activity. In animals receiving IV cisplatin, massive cell damage in the renal medulla and cystic changes within renal tubules were observed, unlike in rats receiving HIPEC. Tumor mitotic activity was lower in morning-treated groups. The median survival of rats in the control group was 8.5 days (95% CI 6.0– 22.0), in HIPEC at ZT0 40.5 days (95% CI 28.0– 47.0, p< 0.001) and in HIPEC at ZT12 32.0 days (95% CI 28.0– 37.0, p< 0.001).Conclusion: In a rat model, ovarian tumor growth disrupted daily variations in intestinal epithelium proliferation and caused genotoxic stress in tumor-free tissues. HIPEC with cisplatin at ZT0 had a better efficacy/toxicity profile than HIPEC with cisplatin at ZT12 and IV administration at both time points.Keywords: ovarian cancer, circadian rhythm, chronochemotherapy, cisplatin, hyperthermic intraperitoneal chemoperfusion, HIPEC, proliferation, DNA damage

The connection between circadian rhythms and cancer is bilateral at the body and cellular level. Negative impact of tumor development on circadian rhythms represents one of its aspects. Materials and methods. 16 FVB/N and 16 FVB/N transgenic HER2/neu female mice were used in the experiment. 4 mice of each line were euthanized at ZT0 (light switch-on time), ZT6, ZT12 and ZT18. Bmal1, Clock and Cry1 gene expression was evaluated in the suprachiasmatic nucleus, liver, mammary tissue and tumors by real-time PCR. Content of BMAL1 and CLOCK proteins in mammary tissue and tumors was evaluated by immunohistochemistry. Results. Absence of clock genes expression and clock proteins content daily oscillations was observed in spontaneous mammary adenocarcinomas of HER2/neu transgenic mice. We also revealed decrease in peak values of clock genes expression in the suprachiasmatic nucleus, liver and mammary gland in tumor- bearing animals. Conclusions. The obtained data confirm the hypothesis about disruption of circadian rhythms in breast tumors and negative influence of tumors on the rhythms of the whole organism.

Introduction: The effects of chemotherapy are known to depend on the time of administration. Circadian rhythms are disturbed in tumors and in tumor bearers. Agents involved in controlling the circadian rhythms (chronobiotics) potentially can modify the outcomes of chemotherapeutics administered at different times of the day. Pineal hormone melatonin (MT) is a prototypic chronobiotic. Objective: The aim of the study was to investigate if MT can affect efficacy or toxicity of chemotherapy drugs administered at the extreme time points of the working day of hospital personnel. Methods: Cyclophosphamide, adriamycin, and 5-fluorouracil (CAF) and adriamycin and docetaxel (AT) cytotoxic drug combinations were administered on day 0 at 11:00 a.m. or at 5:00 p.m. (UTC+03:00) to 6-month-old female HER2/neu transgenic FVB/N mice bearing mammary adenocarcinomas. Some mice were additionally provided with MT in drinking water (20 mg/L) at night 1 week before or 3 weeks after treatment or during both periods. Tumor node sizes, body weight, and blood cell counts were determined right before treatment and on days 2, 7, 14, and 21. Results: Significant decrease in the mean tumor node volume was found by days 14 and 21 upon all CAF and AT treatment schedules, except in animals treated with AT at 5:00 p.m. without supplementation with MT. In the latter case, mean tumor node volume on day 21 was the same as in the control. Supplementation of AT administered at 5:00 p.m. with MT improved the tumor response. CAF and AT regimens supplemented with MT also augmented the number of tumor nodes that did not increase by more than 20% by day 21 as compared to CAF or AT alone, respectively. This effect was significant in groups treated with AT at 5:00 p.m. and consistent upon other schedules. On day 7, leukopenia and anemia were registered in groups treated with CAF regimen; however, blood cell counts normalized by day 14. Both CAF and AT were associated with drop in the body weight registered on day 7. Supplementation with MT did not affect changes of the body weight and blood counts. Conclusions: MT supplementation to cytotoxic drugs can improve antitumor response, especially if it is blunted because of an inappropriate time of administration.

Considering emerging yet contradictory data on circadian rhythms disruption and its effects on tumor initiation and progression, we performed in vivo study to evaluate changes (if any) in clock genes expression in tumor compared to intact tissues as well as to see if tumor development affects normal tissues in tumor-bearing animals. The study was performed in 75 female Wistar rats, intraperitoneally transplanted ovarian cancer was used as a tumor model. Tumor-bearing rats had fragmented circadian rhythmicity of their locomotor activity compared to intact animals. No circadian rhythmicity in proliferation of tumor cells was detected. Precise proliferative rhythmicity was found in normal cells (intestinal epithelium) of intact rats, while significant disruption in such rhythmicity was observed in the same cells of tumor-bearing rats. Average clock genes (BMAL1, CLOCK, CRY1, PER2) expression rate was significantly reduced in tumor cells compared to intact tissues. The data from these experiments let us choosing 2 time points to perform chemotherapy in the following study where effects of chronochemotherapy will be evaluated.

Introduction. Non-steroidal anti-inflammatory drugs and, in particular, diclofenac, are considered as drugs with a potentially anticarcinogenic effect and can be used to enhance the effects of antitumor therapy. Aim. To evaluate the effectiveness of diclofenac as a potential modifier of photodynamic therapy (PDT) in vivo in Ehrlich tumor model in mice. Materials and methods. The study was conducted on male mice of BALB/C strain with intradermally grafted Ehrlich carcinoma. Experimental procedures were performed on day 7 after tumor inoculation. The control group did not receive treatment, in one group only diclofenac was administered, photosensitizer was administered in three groups (photoditazin, 5 mg / kg, intravenously once), tumor was irradiated with a laser (ALOD, 662 nm) or PDT was performed, respectively, and in three groups the procedures indicated above in each case were combined with the administration of diclofenac. 2 series of experiments were conducted to assess the reproducibility of the results. The size of the tumor was recorded after exposure. Results. In the first series of the experiment, in all groups with the administration of diclofenac (45 mg/kg/day), a high animal mortality was unexpectedly observed, associated with the toxic effect of diclofenac, which required a change in its administration regime and dose (15 mg/kg) in the second series of the experiment. The administration of diclofenac did not significantly affect the efficacy of PDT, and also did not have a modifying effect in other groups (with the introduction of a photosensitizer or laser irradiation) compared with similar groups without diclofenac. PDT with photoditazine turned out to be the most effective treatment regimen: in 8 out of 10 mice in the group from the 21th day after procedure the tumors were not recorded, the animals were alive until the end of the observation for 60 days. The introduction of a photosensitizer and radiation without a photosensitizer did not lead to a significant inhibition of tumor growth. Conclusion. In vivo on a model of intradermally grafted Ehrlich tumor in mice, diclofenac does not have a modifying effect on the antitumor effects of PDT.

Circadian rhythms and tumor development are interconnected as the factors like light pollution which disrupt circadian rhythms increase the risk of cancer, and oncological diseases are associated with changes in organism’s circadian rhythms. Circadian changes in intestinal epithelium and mammary tumors proliferation and apoptosis in HER-2/neu overexpressing FVB/N mice and assessment of melatonin’s influence on these parameters were studied in this work. It was shown by us that intestinal epithelium in mice exhibits circadian rhythm of proliferation with the peak in the morning and in tumor-bearing mice this rhythm is disrupted. Exogenous melatonin contributes to circadian rhythm of intestinal epithelium proliferation. Circadian changes in mammary tumors proliferation rate depend on melatonin secretion or supplementation time. Thus, melatonin may be considered as a perspective drug in anticancer therapy modulating circadian rhythms in cancerous and normal tissues.

Identification of molecular targets and mechanism of action is always a challenge, in particular - for natural compounds due to inherent chemical complexity. BP-Cx-1 is a water-soluble modification of hydrolyzed lignin used as the platform for a portfolio of innovative pharmacological products aimed for therapy and supportive care of oncological patients. The present study describes a new approach, which combines in vitro screening of potential molecular targets for BP-Cx-1 using Diversity Profile - P9 panel by Eurofins Cerep (France) with a search of possible active components in silico in ChEMBL - manually curated chemical database of bioactive molecules with drug-like properties. The results of diversity assay demonstrate that BP-Cx-1 has multiple biological effects on neurotransmitters receptors, ligand-gated ion channels and transporters. Of particular importance is that the major part of identified molecular targets are involved in modulation of inflammation and immune response and might be related to tumorigenesis. Characterization of molecular composition of BP-Cx-1 with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and subsequent identification of possible active components by searching for molecular matches in silico in ChEMBL indicated polyphenolic components, nominally, flavonoids, sapogenins, phenanthrenes, as the major carriers of biological activity of BP-Cx-1. In vitro and in silico target screening yielded overlapping lists of proteins: adenosine receptors, dopamine receptor DRD4, glucocorticoid receptor, serotonin receptor 5-HT1, prostaglandin receptors, muscarinic cholinergic receptor, GABAA receptor. The pleiotropic molecular activities of polyphenolic components are beneficial in treatment of multifactorial disorders such as diseases associated with chronic inflammation and cancer.

Chemotherapeutic drugs negatively influence on normal tissues with high proliferative activity such as intestinal epithelium. The damage of chemotherapy to healthy tissues significantly reduces oncological patients’ quality of life. Drugs based on polyphenolic natural compounds are a perspective mean of adjuvant therapy due to their antioxidant and antiinflammatory properties. In this study we have showed that a single intragastric administration of BPC3 polyphenolic composition 2 hours before 5-fluorouracil treatment alleviates small intestine damage in C57Bl/6 mice after 3.5, 24 or 72 hours. BPC3 effect manifested in reduced apoptosis rate, increased mitotic activity of the epithelium in the regenerative phase and protection of LGR5-positive jejunum stem cells against 5-fluorouracil induced damage.

The antitumor activity of the antifibrinolytic drug ε-aminocaproic acid (ACC) in the therapeutic form (5% ACC solution in physiological solution instead of drinking water) was studied in the ascitic and solid Ehrlich tumor. ACC did not affect the growth of ascitic, as well as a solid Ehrlich tumor, when the drug was administered 72 hours after the tumor inoculation. At the same time, when it was administered 7 days after tumor inoculation, the drug had a statistically significant antitumor effect on the tumor nodes formed (8-10 mm in diameter). ACC antitumor effect allows to assume that it influences tumor stroma rather than the parenchyma. This effect is not fully understood and needs further study.

For the first time comparative study of open and closed techniques of hyperthermic intraperitoneal chemoperfusion (HIPEC) in terms of safety and efficacy was performed in a rat model of ascetic ovarian cancer transplanted intraperitoneally. Original device and implantation technique of the device into peritoneal cavity were developed and used in the study. All animals after tumor implantation were randomized into 4 groups: 1 - control, intraperitoneal administration of 0.5 ml of saline (n=19); 2 - closed HIPEC with cisplatin, 20 mg/ kg (n=15); 3 - open HIPEC with cisplatin, 16 mg/kg (n=16); 4 - open HIPEC with mitomycin C (n=10). In preliminary stage while working out the original open technique of HIPEC we established that it requires dose reduction for cisplatin from 20 to 16 mg/kg compared to closed technique of HIPEC. We didn't find significant difference between groups 1, 2 and 3 in terms of number of postoperative complications. According to the results of analysis of body weight changes in postoperative period open HIPEC was worse tolerated compared to close HIPEC. Median survival of rats in group 2 was 53 days which was significantly higher compared to median survival in group 2-25 days (p=0,044). Open HIPEC with mitomycin C turned out to be less effective than open HIPEC with cis-platin but equally effective compared to closed HIPEC with cisplatin. However this is preliminary result and more rats have to be included in group 4 in order to make confident conclusion. Overall open HIPEC with cisplatin in accordance with suggested technique seem to be more promising than closed HIPEC in terms of improving outcomes of patients with peritoneal carcinomatosis.

This study aimed to evaluate the effect of lignin-derived polyphenolic composition BP-C3 on the efficacy and hematological toxicity of cyclophosphamide (CPA). Male and female Swiss-H derived mice bearing benzo[a]pyrene-induced soft tissue sarcomas were treated with CPA 300 mg/kg, BP-C3 75 mg/kg, or a combination. Tumor growth inhibition in male mice treated with CPA, BP-C3, or a combination of CPA and BP-C3 was significant and corresponded to 78%, 45%, and 82%, respectively, on day 21 after CPA administration on day 0. In female mice, tumor growth inhibition was 58%, −11%, and 35% when treated with CPA, BP-C3, or a combination of CPA and BP-C3, respectively. CPA administration resulted in significant hematological toxicity evidenced by a decreased white blood cell count on day 4 (2.43 ± 1.77 × 109/L in male mice and 1.19 ± 0.71 × 109/L in female mice) and anemia development on day 7 (6.55 ± 1.74 × 1012/L in male mice and 5.89 ± 2.24 × 1012/L in female mice). The red blood cell count measured on day 7 in animals treated with the combination of BP-C3 and CPA constituted 7.12 ± 1.17 × 1012/L and 7.36 ± 2.07 × 1012/L for male and female mice, respectively. The results of our study demonstrate the antitumor activity of BP-C3 in male mice bearing soft tissue sarcomas. Neither the antitumor activity nor the hematological toxicity of CPA were significantly influenced by BP-C3. A less pronounced effect of CPA on RBC count is demonstrated when this agent is given jointly with BP-C3.

BP-C3 is a formulation, which comprises lignin-derived polyphenolic composition of benzenepolycarboxylic acids (BP-Cx-1) with iron complex, selenium, ascorbic acid and retinol, and possesses geroprotective activity. The present study examined the effect of BP-C3 (80 mg/kg, administered 18 times in total by gavage) on the development of haematological and intestinal manifestations of toxicity following 5-fluorouracil (5-FU; 150 mg/kg, administered once via intravenous injection) administration in outbred male Swiss-H Rappolovo (SHR) mice. The use of BP-C3 on therapeutic and preventative/therapeutic schedules demonstrated that it was protective against the toxic effect of 5-FU exerted on the lymphopoietic organs. Administering ВР-С3 24 h after 5-FU (therapeutic schedule) had an effect on the recovery of leukopoiesis and prevented anaemia in the mice. In the mice that received 5-FU and 5-FU with BP-C3 prior to and following administration of the chemotherapeutic agent (preventative/therapeutic schedule), mild anaemia developed by day 7. Administration of BP-C3 without 5-FU did not affect blood cell differentiation in the mice. Thus, BP-C3, depending on the administration schedule, had different effects on the haematological parameters of haematopoietic organs and peripheral blood in mice exposed to 5-FU. BP-C3 promoted intestinal crypt survival when administered on the preventative/therapeutic and therapeutic schedules, suggesting that the formulation protects the epithelium of the small intestine against damage by 5-FU.

There remains an unmet medical need for radioprotective and mitigative agents. BP-C2 is a novel lignin-derived polyphenolic composition with ammonium molybdate, developed as radioprotector/radiomitigator.The present study evaluated BP-C2 for the mitigation of acute radiation syndrome (ARS).A total-body irradiation mouse model (TBI, 4.0-8.0 Gy) was used in the study.In a 30-day survival study, performed in CBA mice, BP-C2, at a dosage of 81.0 mg/kg, improved survival (dose reduction factor (DRF) = 1.1) and increased the formation of endogenous spleen colony-forming units (CFU). In C57BL/6 mice, BP-C2, when administered daily for 7 days, starting 24 hours after TBI, also improved survival. In animals irradiated with 5.0 Gy, BP-C2 increased the number of CFUs (6.7 ± 5.1) compared to the 5.0 Gy placebo group (2.3 ± 2.3, p = .0245). The number of surviving intestinal crypts was maintained in the 5.0 Gy BP-C2 group (133.7 ± 13.9), in contrast to the 5.0 Gy placebo group (124.2 ± 10.5, p .0023). BP-C2 also increased the number of LGR5 + positive cells in intestinal crypts.BP-C2 mitigates radiation-induced damage in mid-lethal range of radiation doses. Effects are mediated by enhancement of extramedullar hematopoiesis in the spleen and a protective effect on the intestinal epithelium.

A systematic review of the studies where HIPEC combined with cytoreductive surgery was used in patients with primary advanced ovarian cancer was performed to understand is there a role for this treatment modality not only in recurrent but in primary advanced ovarian cancer. The results are controversial but there is a strong trend for improvement of the long-term outcomes of patients with primary advanced ovarian cancer after HIPEC.

The study or antitumor effects of dioxadet, cisplatin, melphalan, paclitaxel, mitomycin C, cyclophosphamide and gemcitabine at intraperitoneal (i.p.) and intravenous (i.v.) administration as monochemotherapy and polychemotherapy in a rat model of ascitic ovarian cancer was carried out in 244 female Wistar rats. Ovarian cancer was transplanted i.p. at a number of 1 x 10(7) tumor cells. The drugs were administered once in 48 hours after ovarian cancer transplantation i.p. or i.v. for monotherapy--in maximum tolerated doses, for i.p. polychemotherapy--in half doses from maximum tolerated doses. Antitumor effects of the treatment were estimated in increase in median survival time (MST) compared to control rats who were administered saline i.p. At i.p. administration dioxadet, cisplatin and melphalan increased MST by 79%, 88% and 144%, respectively, while at i.v. administration these drugs didn't affect MST. Mitomycin C and paclitaxel had stronger antitumor action at i.v. administration increasing MST by 152% and 81%, respectively, while at i.p. administration these drugs increased MST by 35 and 45%, respectively. Combinations dioxadet + cisplatin, dioxadet + cyclophosphamide and dioxadet + paclitaxel at i.p. administration increased MST by 305%, 277% and 133%, respectively, and had additive antitumor action compared to mono-effects of these drugs. Gemcitabine and combination dioxadet + gemcitabine at i.p. administration didn't significantly affect survival of rats with ovarian cancer. Intraperitoneal monochemotherapy and polychemotherapy could be more effective in the treatment of peritoneal carcinomatosis from ovarian cancer compared to systemic administration of the drugs.