The influence of an enamine usnic acid derivative (a tyrosyl‑DNA phosphodiesterase 1 inhibitor) on the therapeutic effect of topotecan against transplanted tumors in vivo
V. P. Nikolin1 · N. A. Popova1,4 · V. I. Kaledin1 · O. A. Luzina2 · A. L. Zakharenko3 · N. F. Salakhutdinov2,4 · O. I. Lavrik3,4,5
Received: 22 January 2021 / Accepted: 23 July 2021
© The Author(s), under exclusive licence to Springer Nature B.V. 2021
Abstract
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a repair enzyme for 3′-end DNA lesions, predominantly stalled DNA–topoi- somerase 1 (Top1) cleavage complexes. Tdp1 is a promising target for anticancer therapy based on DNA damage caused by Top1 poisoning. Earlier, we have reported about usnic acid enamine derivatives that are Tdp1 inhibitors sensitizing tumor cells to the action of Top1 poison (Zakharenko in J Nat Prod 79:2961−2967, 2016). In the present work, we showed a sensitizing effect of an enamine derivative of usnic acid (when administered intragastrically) on Lewis lung carcinoma in mice in combination with topotecan (TPT, Top1 poison used in the clinic). In the presence of the usnic acid derivative, both the volume of the primary tumor and the number of metastases significantly diminished. The absence of acute toxicity of this compound was demonstrated, as was the importance of the method of its administration for the manifestation of the sensitizing properties.
Keywords DNA repair enzymes · Tdp1 inhibitor · Lewis lung carcinoma · Topotecan · Enamine usnic acid derivative
Abbreviations TPT Topotecan UA Usnic acid
Introduction
The antitumor effect of a number of anticancer drugs is based on impairing the ability of topoisomerases (Top) I and II to control DNA supercoiling and relaxation, which occur during the main processes of DNA metabolism. Such compounds include the well-known cytostatics camptoth-ecin (and its derivatives topotecan (TPT) and irinotecan) Kaledin—deceased.
* O. I. Lavrik [email protected]
1 Institute of Cytology and Genetics, Siberian Branch
of Russian Academy of Sciences, 10 Akademika Lavrentieva Ave., Novosibirsk, Russian Federation 630090
2 N. N. Vorozhtsov Institute of Organic Chemistry, Siberian Branch of Russian Academy of Sciences, 9 Akademika Lavrentieva Ave., Novosibirsk, Russian Federation 630090
3 Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy
of Sciences, 8 Akademika Lavrentieva Ave., Novosibirsk, Russian Federation 630090
4 Novosibirsk State University, 1 Pirogova Str., Novosibirsk, Russian Federation 630090
5 Altai State University, 61 Lenina Ave., Barnaul, Russian Federation 656049
and etoposide. DNA repair enzyme activity is one of the reasons for tumor resistance to the action of DNA-damaging agents, thereby weakening their therapeutic effect [1, 2]. Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is one of these enzymes that play a key role in the removal of DNA dam- ages resulting from inhibition of Top1 by camptothecin and its derivatives [3].
It has been suggested that Tdp1 inhibi- tion increases the therapeutic effect of camptothecin deriva- tives and other DNA-damaging agents [4]. The hypothesis that Tdp1 is responsible for drug resistance in some cancers is supported by a number of studies: Tdp1 deficiency in Tdp1 knockout mice and in human cell lines with a SCAN1 mutation (that reduces the activity of this enzyme) leads to hypersensitivity to camptothecin or its derivatives [5–7]. Besides, suppression of Tdp1 expression with minocycline enhances the antimetastatic effect of irinotecan and increases the lifespan of experimental animals [8]. Conversely, in cellswith increased Tdp1 expression, camptothecin causes less DNA damage [9, 10]. Moreover, in intestinal tumors with overexpression of Tdp1, the response to irinotecan therapy is smaller [11]. These data suggest that a combination of anti- cancer drugs and Tdp1 inhibitors can significantly increase the effectiveness of chemotherapy.
The number of Tdp1 inhibitors described in the literature is quite large; they belong to different chemical classes, most are derivatives of natural biologically active compounds, and have an inhibitory effect in the range of 0.018–10.000 μM reviewed by [12–14], also described in original articles [15–26]. There are also studies on the synthesis of dual inhibitors (acting on Top1 and Tdp1) that suppress Tdp1 activity in the micromolar concentration range [19, 27–33]. In this case, a synergistic effect is expected from the inhibi- tion of these enzymes by the same molecule, and this action can decrease the toxic effects of therapy. At the same time, it may be difficult to choose an individual dose for patients with different genetic backgrounds.
Recently, the attention of researchers was attracted by a natural compound of lichen origin, usnic acid (UA, Fig. 1), and it was shown that the most striking examples of ther- apeutic applications of UA and derivatives are related to overcoming the resistance of bacterial infections and tumor tissues to known drugs [34]. The mechanism of this action is not fully clarified and is probably linked to the inhibi- tory effect of UA and its derivatives on repair enzymes: poly(ADP-ribose) polymerase 1 (PARP-1), DNA polymer- ase β (pol β), and Tdp1 [15, 35].
Previously [36], we demonstrated that enamine deriva- tives of (+)-UA are quite effective inhibitors of Tdp1, have low toxicity to human breast adenocarcinoma cell line MCF- 7, and enhance the cytotoxic properties of camptothecin in vitro. In that study, we synthesized compounds of the enamine type from (+)-UA via a reaction with aliphatic and aromatic amines. The most pronounced synergistic effect (10.5-fold enhancement) was shown by OL9-116 (Fig. 1) when combined with camptothecin in experiments on MCF-7 cells; this compound was obtained by the reaction of (+)-UA with 4-(3-aminopropyl)-2,6-di-tert-butylphenol. The half-maximal inhibitory concentration (IC50) of OL9- 116 against Tdp1 was found to be 0.16 μM.
Earlier [37], we studied the antimetastatic activity of TPT separately and in combination with OL9-116 in a Lewis lung carcinoma (LLC) model. In this work, we assessed the effect of a drug combination on the number, size, and location of experimental metastases. The most pronounced antimeta- static effect was observed during combined use of TPT and OL9-116. In that study, this phenomenon was confirmed both by a macroscopic examination of lungs (a decrease in the number of metastases, inhibition of metastasis up to 98%) and by morphological and morphometric analyses of lung sections (an increase in the index of inhibition of metas- tases’ size by 63–86% depending on the OL9-116 dose). In the control group, and in the groups of animals that received only one of the drugs, metastases were located subcapsular, peribronchial and perivascular, while in the groups of com- bined therapy, predominantly perivascular.
In the present work, we investigated the ability of OL9- 116 to enhance the effect of TPT (a camptothecin deriva- tive used in the clinic) in two models of transplanted mouse tumors in vivo. We also chose the optimal method of admin- istration of the Tdp1 inhibitor and clarified the mechanism of the antimetastatic action of combination TPT + OL9-116 (direct impact on the cells of lung metastases after their separation from the primary tumor).
Materials and methods
Chemicals
The work was carried out using the following reagents: TPT (ACTAVIS GROUP PTC ehf., Bucharest, Romania), dime- thyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA), and Tween 80 (Panreac, Barcelona, Spain). Tdp1 inhibitor OL9- 116 was synthesized as described in our previous work [36].
Animals and tumors
Three- to four month-old male and female BALB/C, CBA/ Lac/sto (CBA), and C57BL/6Mv (C57BL) mice from the breeding colony of the Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences, were used in the study. They were kept on sawdust in plastic cages and fed a standard diet (Laboratorkorm, St. Petersburg, Structures of (+)-usnic acid and its derivative OL9-116
cancer cell lines were obtained from the cell depository ofthe Institute of Cytology and Genetics (Novosibirsk, Rus- sia) and are maintained in mice as a transplanted tumors. Mouse model of LLC is the most widely used lung cancer model of the same origin, LLC cell line maintains high tumorigenicity and lung metastasis in C57BL mice [38, 39], and subcutaneous LLC transplantation leads to the appearance of lung metastases on days 17–21 [40].
Krebs-2 carcinoma is nonspecific in its host require- ments and can be propagated in mice of various genetic constitutions. Krebs-2 is obtained from epithelial cells of the abdominal wall of a mouse. With intraperitoneal inoculation, it forms an ascites form. The tumor is weakly immunogenic for mice of all strains. Does not metastasize [41–44].
Krebs-2 tumors were maintained in ascitic form in BALB/c or CBA mice, but LLC was maintained in solid form after subcutaneous transplantation in C57BL mice. In the latter case, prior to the transplantation, the tumors were minced by means of scissors and passed through a sieve 0.914 mm in diameter. The tumor tissues were resuspended in a 0.9% NaCl solution and injected either intramuscularly (in a volume of 0.1 ml) or intravenously. Ascites tumors were transplanted similarly.
The animals were treated with TPT and Tpd1 inhibitor OL9-116 and were evaluated one to several days after the tumor transplantation (see the experimental details below). TPT was always administered intraperitoneally (i/p), but Tdp1 inhibitor OL9-116 was given in a DMSO–Tween 80 suspension (0.3 ml/mouse) intragastrically (i/g) or injected i/p. Both administration routes were employed simultaneously. In different experiments, the animals were euthanized 8–18 days after the transplantation of tumor cells. Control groups (no treatment) received vehicle (DMSO–Tween 80 suspension).
We started the treatment on the 1–3rd day after the tumor was inoculated, in the expectation that it had just begun to form. The end point was considered the death of mice (experiment to determine the life expectancy) or a day or two before the estimated time of death of mice in the control.
Evaluation of the antitumor effect of the drugs was based on the size and weight of the LLC solid tumors, their daily gain in volume, survival time of the mice, and the number of tumor metastases in the lungs. For estima- tion of the daily gain in LLC tumor volume, the tumor nodules were periodically measured with a caliper. Metas- tases in the lungs were counted microscopically (biologi- cal research microscope MBI-3, LOMO, St. Petersburg, USSR) at threefold magnification after their fixation in 10% formalin. The weight of Krebs-2 ascites was assessed by the difference in the weight of mice before and after removal of ascites. The number of tumor cells in ascites was determined using a Goryaev camera.
Statistical analysis
The measurements and calculation of the primary data (tumor size and metastasis number) were conducted in a blinded manner. The animal data were statistically evalu- ated by one-way ANOVA. Post hoc assessment was per- formed using Tukey’s honestly significant difference (HSD) test. Data with p < 0.05 were considered statistically sig- nificant. Statistical package STATISTICA version 12.5 was utilized for this analysis, and all results are expressed as mean ± SEM.
Results
Acute toxicity
The general toxic effect of OL9-116 was evaluated in intact C57BL male mice by means of the change in body weight after a single i/g administration of various doses of OL9-116 in combination with i/p administration of TPT. The data are presented in Fig. 2. In Fig. 2, readers can see that the applied dose of TPT, when administered i/p, did not affect the weight of the animals. Simultaneous i/g administration of OL9-116 at doses of 80 and 160 mg/kg did not lead to a decrease in body weight, and only the OL9-116 dose of 240 mg/kg (green graph) in combination with TPT caused a slight weight decrease, not more than 5%. OL9-116 alone at this dose (orange graph) showed no toxicity either.
Single oral administration of OL9-116 at doses of 80–240 mg/kg was well tolerated by the mice without any signs of toxicity or discomfort. During the assessment of the Dynamics of body weight of mice after a single TPT injection at a dose of 2 mg/kg in combination with various doses of OL9-116. TPT was injected i/p, and OL9-116 i/g; control: vehicletolerance to treatment, the state and behavior of the animals were taken into account, as was the body weight and the absence of deaths among the animals. The state and behavior of mice were without any peculiarity. Mobility, interest in food and water, presence / absence of sleepiness and slug- gishness, etc. were assessed. It was concluded that the toler- ability of OL9-116 monotherapy and of its combination with TPT is satisfactory.
The TPT dose
First, we studied the impact of OL9-116 on the antitumor effect of TPT on the ascites version of the Krebs-2 tumor, which enables direct access of the chemotherapy drug to tumor cells. For this purpose, several preliminary experi- ments were conducted to determine the suitable range of TPT doses. The experiments were performed on BALB/c female mice, which were injected i/p with 2 million ascites tumor cells on day zero. After 1, 2, and 3 days, the experi- mental mice were also injected with TPT i/p in a single dose of 1.3 mg/kg. The mice were weighed daily until all con- trol (TPT-free) animals died naturally as a result of tumor growth. This happened between 19 and 21 days after the tumor transplantation. As for the mice that received TPT at the maximum dose 1.3 mg/kg × 3, ascites was not detected in them throughout the observation period. They were with- drawn from the experiment and euthanized 25 days after the tumor inoculation. Lifespan data are shown in Table 1. The results showed that the dose and treatment regimen used were not suitable for studying the influence of Tdp1 inhibi- tors on the antitumor effect of TPT. Therefore, subsequently, we administered TPT one to two times to transplanted- tumor–bearing mice in a smaller dose not exceeding 1 mg/ kg in total and 0.75 mg/kg single dose.
A significant increase in the average lifespan (by 65%)
was observed after triple administration of TPT in a dose of 0.5 mg/kg. Double administration of TPT increased the lifespan by only 30%.
Thus, single administration of TPT at 0.5 mg/kg is suf- ficient to reveal the potentiating effect of OL9-116.
The influence of TPT and OL9‑116 on the growth of the ascites Krebs‑2 tumor
In the following experiment, the effect of TPT in combina- tion with OL9-116 was studied, depending on the method of administration of OL9-116.
TPT was injected i/p 1 day after 2 million tumor cells were transplanted i/p. The TPT dose in this experiment was 0.75 mg/kg. Simultaneously with TPT, OL9-116 was administered once i/g or i/p to the animals at 80 mg/kg. As presented in Fig. 3a, by the time of experiment termination, in the group of mice receiving only TPT (group 2), tumor growth inhibition (TGI) was 69% as compared with vehi- cle-treatment control (group 1), whereas in mice receiving TPT and OL9-116 i/g (group 3), TGI was 77%. Taking into account the number of tumor cells in the mice, TGI values were 79% under the action of TPT alone and when TPT was combined with the Tdp1 inhibitor (Fig. 3b).
When comparing groups 1 and 5, we noted that when administered i/g, OL9-116 itself (80 mg/kg) does not affect tumor growth, but when administered i/p, it significantly reduces both the tumor weight (Fig. 3a) and the number of tumor cells (Fig. 3b) as compared to the control (groups 1 and 6). In this case, the combined use of OL9-116 (i/p administration, 80 mg/kg) with TPT (0.75 mg/kg, i/p) led to an antagonistic effect (the effectiveness of both was reduced from 69% for TPT and 48% for OL9-116 to 37% for combi- nation). When administered i/g, OL9-116 (80 mg/kg) had no effect either together with TPT or by itself. After i/g admin- istration of OL9-116 against the background of TPT treat- ment, the effect of the latter did not increase significantly (from 69 to 77%, groups 2 and 3 respectively).
Therefore, the use of OL9-116 (80 mg/kg) i/g revealed its propensity to enhance the antitumor effect of TPT; however, the differences were not significant. Accordingly, in the next experiment, we increased the magnitude of the effect on mice by means of both TPT and OL9-116. In this setup, the drugs were administered twice, at 24 and 48 h after tumor transplantation, and the total dose of TPT 1 mg/kg was a quarter higher (at a single dose
Median 25%-75%
Non-Outlier Range
Krebs-2 tumor in male and female CBA mice equally after i/p admin- istration of TPT and i/g administration of OL9-116. *Significantly differs from the weight of ascites in groups 1 (vehicle) and 2 (TPT only), p < 0.005 and p = 0.05, respectively. The numbers indicate the average means ± SEM, below them TGI in percent. All four groups consisted of 6 mice each. The treatment was carried out two times at 24 and 48 h after tumor transplantation, single doses were 0.5 mg/kg for TPT and 80 mg/kg for OL9-116
The influence of TPT and OL9‑116 on the growth and metastasis of LLCIn the next series of experiments, a different experimental model was tested, namely transplantable LLC in C57BLThe influence of TPT and OL9-116 on the growth of an ascites Krebs-2 tumor in male CBA mice after intraperitoneal (i/p) admin- istration of TPT and i/p or intragastric (i/g) administration of OL9-
116. The numbers indicate the average means ± SEM, below them TGI in percent. 1—Control (vehicle), 6 mice; 2—TPT, 5 mice; 3— TPT + OL9-116 i/g, 6 mice;4—TPT + OL9-116 i/p, 6 mice; 5— OL9-116 i/g, 4 mice; 6—OL9-116 i/p, 4 mice. For all groups TPT dose 0.75 mg/kg, OL9-116 dose 80 mg/kg. a The average weight of ascites; *p = 0.02 as compared with group 1. b The average number of tumor cells in ascites; **p = 0.04 as compared with group 1of 0.5 mg/kg), while the total dose of the Tdp1 inhibitor was doubled to 160 mg/kg. Figure 4 shows that OL9-116 increased the effectiveness of therapy threefold, from 0.83 to 0.23 g (judging by the weight of ascites; due to the small volume of ascites, we could not determine its cel- lular concentration).
Thus, the results indicated that under the conditions of direct contact of TPT with tumor cells, its therapeutic effect is significantly enhanced by the simultaneous i/g administration of the Tdp1 inhibitor.mice. After intramuscular transplantation, the tumor grows in the form of solid nodes at the site of transplantation and yields multiple metastases to the lungs [45]. In this experi- ment, the effectiveness of the therapy was assessed by means of the weight of solid tumors and the metastasis number in the lungs of mice euthanized 18 days after the tumor trans- plantation. On days 4, 5, and 6, mice were treated as fol- lows: group 1 with 0.3 ml of a vehicle (DMSO–Tween 80 emulsion) i/g; mice of groups 2 and 3 were injected with TPT (i/p) at a single dose of 2 mg/kg and simultaneously with either vehicle i/g (group 2) or OL9-116 (80 mg/kg i/g) in 0.3 ml of a vehicle (group 3); group 4—with OL9-116 (80 mg/kg i/g). The mice were euthanized on day 18 after the tumor transplantation. Figure 5 shows data on the growth of solid tumors in this experiment, depending on the effect of TPT or TPT with OL9-116, and Fig. 6 presents the average weight of solid tumors (6a) and the number of metastases (6b) in the lungs at the end of the experiment.After TPT monotherapy, TGI of the solid tumors was only 22% as compared to the control, whereas the combi- nation of TPT with OL9-116 resulted in 1.8 suppressionDynamics of LLC tumor growth in male and female C57BL mice. Black squares: the control group (vehicle), red circles: TPT only (i/p), blue triangles: TPT in combination with OL9-116 (i/g), magenta triangles: OL9-116 (i/g). The treatment was carried out three times on days 4, 5 and 6, single doses were 2 mg/kg for TPT and 80 mg/kg for OL9-116. (Color figure online)
(b) 60 of tumor growth (Fig. 6a). In terms of the influence on metastases, the effect of treatment was much more pro- nounced: TPT alone reduced their number threefold, whereas TPT with OL9-116 by more than 90% as com- pared with the control (Fig. 6b).
Naexperiment, we transplanted LLC tumor cells intrave- nously in C57BL male mice, i.e., generated artificial lung metastases without a primary tumor. Concurrently with a total dose of TPT of 2 mg/kg, the animals were injected with OL9-116 (i/g) at a total dose of 120 mg/kg. The drug administration was carried out twice: 3 and 5 days after the tumor transplantation. The mice were euthanized on day 18. The results are depicted in Fig. 7. As illustrated in the figure, the group of mice treated with OL9-116 alone did not differ from the control group in the average number of metastases per mouse. TPT under the condi- tions of this experiment reduced the number of metastases from 37 to 8 as compared with control, whereas TPT in combination with OL9-116 from 37 to 3.5 as compared with the control and from 8 to 3.5 as compared with the TPT group.
Thus, the antimetastatic effect of OL9-116 against the background of TPT treatment is related to the direct impact of this combination on the cells of lung metastases.
The influence of TPT and OL9-116 on the growth and metas- tasis of LLC in male and female C57BL mice. All four groups con- sisted of 6 mice each. The treatment was carried out three times on days 4, 5 and 6, single doses were 2 mg/kg for TPT and 80 mg/kg for OL9-116. a The average weight of a primary tumor in mice with subcutaneously transplanted LLC. The numbers indicate the average means ± SEM, below them TGI in percent. p = 0.0047 for groups TPT and TPT + OL9-116. b The number of lung metastases in mice on day 18 after tumor transplantation. The numbers indicate the average means ± SEM, below them TGI in percent. p = 0.0003 for groups TPT and TPT + OL9-116
Discussion
According to information from the Register of Toxic Effects of Chemical Substances (RTECS; a database on toxicity of substances), oral toxicity (50% lethal dose; LD50) of (+)-UA for mice is 836 mg/kg (75 mg/kg for subcutaneous adminis- tration and 25 mg/kg for intravenous administration). Some modifications of the UA backbone lead to a two to fourfold decrease in cytotoxicity [46]. There are no in vivo data on
The average number of lung metastases per mouse (C57BL
sensitizing effect with any route of administration (Fig. 3). On the other hand, a double dose of the drug injected i/g had a statistically significant sensitizing effect on tumor growth (Fig. 4).
Thus, it can be assumed that for the sensitization to occur, the drug must pass through the gastrointestinal tract and probably should be metabolized. To elucidate the pos- sible transformations of the compound in the liver, separate studies are required. The intrinsic antitumor effect after i/p administration may proceed via a different mechanism not associated with the repair of DNA damage caused by topoi- somerase 1. For example, the effect of UA on the phospho- rylation site of suppressor of malignant tumor emergence p53 (Ser20 instead of Ser15 or Ser46) and, thus, on cell sur- vival has been reported [51]. In the same work, it was shown that necrosis was observed in the cell line with active p53 (Capan-2) after incubation with UA for 48 h but not in the cell line with inactive p53 (T-47D) with the same treatmentmale). Control group (vehicle)—14 mice, TPT group—15 mice, TPT + OL9-116 13 mice, OL9-116 6 mice. The treatment was car- ried out two times on days 3 and 5, single doses were 1 mg/kg for TPT and 60 mg/kg for OL9-116. p = 0.002 for groups TPT and TPT + OL9-116
the toxicity of UA derivatives, except for the hydrazinothia- zole derivative with a substituent on ring A [47]: LD50 is more than 5000 mg/kg. In addition, when choosing a dose, one should take into account the lack of information on the bioavailability of UA derivatives. According to a study on the pharmacokinetics of a sodium salt of (+)-UA, i.e., the so-called soluble form, the bioavailability is 78% [48]. With the introduction of the enamine substituent, the hydrophilic- ity of the molecule increases, but there are no data on the bioavailability of such UA derivatives. Consequently, we investigated the effect of stand-alone 80–240 mg/kg doses via i/g administration. These are high enough but presuma- bly safe for i/g administration. Indeed, neither Tdp1 inhibitor OL9-116 alone nor its combination with TPT resulted in a weight loss, behavior change, or death after i/g administra- tion (Fig. 2).
The i/p route is considered analogous to intravenous administration for small animals [49], but when choosing a dose for i/p administration, we kept in mind that systemic action is mediated by the primary route of absorption: mes- enteric vessels, which drain into the portal vein and pass through the liver [50]. We used i/g administration as analo- gous to oral administration in humans. Gastric gavage is often used in research settings, instead of mixing substances in water or food, to ensure precise and accurate dosing of animals [49]. For a comparison of the i/p and i/g methods of administration, we chose a dose of 80 mg/kg. At this dose, the compound exerted its own antitumor activity when administered i/p, but not i/g, and did not have the desired
Besides that, p53 production strongly increased after the first 6 h of the exposure of A2780 tumor cells to UA, which can also affect cell survival [52]. In addition to its effect on the tumor suppressor p53, UA also causes oxidative stress and mitochondrial dysfunction [53, 54]. A number of other pos- sible mechanisms of the cytotoxic effect of UA have been reported (reviewed in [55]). We assume that derivatives of UA may act in a similar way.
Metastasis is a more significant problem than the primary tumor owing to the unpredictability of the lesion appear- ance and to disruption of the functioning of various organs and systems. We studied the influence of OL9-116 after i/g administration on the antitumor and antimetastatic activ- ity of TPT using LLC in mice as an example. As shown in Fig. 6b, the Tdp1 inhibitor in combination with TPT sta- tistically significantly reduces the number of metastases as compared to TPT alone. It was unclear what increased the antimetastatic impact of the applied treatment: the effect on the primary tumor, which produced fewer metastases, or direct action of the drugs in the lungs on the metastatic cells after their separation from the primary tumor. To answer this question, we took advantage of the fact that intravenous administration of LLC yields metastases in the lungs with- out the formation of a primary node. According to the data presented in Fig. 7, the drug combination acts directly on lung metastases. Morphological and morphometric analysis of lung sections [37] confirms the conclusion that the com- bined use of OL9-116 and TPT causes the most pronounced antimetastatic effect: there was a decrease in the frequency of metastasis, significant reduction in the average area of metastases, their perivascular position (as opposed to sub- capsular, peribronchial and perivascular in the groups that received the drugs separately and in the control).
The results showed that when TPT is introduced into anascites Krebs-2 tumor, its therapeutic effect is significantlyenhanced by simultaneous treatment with Tdp1 inhibitor OL9-116. It was established that the latter manifests a pro- nounced sensitizing activity when administered i.g. simul- taneously with TPT. With respect to LLC, the combination of OL9-116 and TPT has a pronounced synergistic effect on both TGI and the number of lung metastases. It was found that the antimetastatic effect of OL9-116 against the back- ground of TPT treatment is attributable to the direct impact of this combination on the cells of lung metastases.
The modern literature contains a significant number of studies devoted to the development of Tdp1 inhibitors and their ability to sensitize tumor cells to the action of Top1 poisons, but almost all of them is performed in vitro (reviewed in [14, 56]). The only exception is our three works describing the antitumor and antimetastatic effect of another UA derivative [20, 57] and arylcoumarin derivative [22] in mice. These studies demonstrate the effectiveness of Tdp1 inhibitors as sensitizers of mouse tumor models to TPT. The next step towards the clinical use of our data will be preclini- cal studies of such combinations.
Acknowledgements This work was funded by a Grant from the Min- istry of Science and Higher Education Russian Federation (Agreement No. 075-15-2020-773).
Data availability All data generated or analysed during this study are available on request from the authors.
Declarations
Conflict of interest The authors declare no conflicts of interest.
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