An extensive literature review was performed using the PubMed database in order to identify studies involving the anticancer properties of quinolones and their derivatives. The PubMed database was searched using the following terms: anticancer activity of quinolones, bladder cancer, prostate cancer, cancers of the genitourinary system, and treatment of genitourinary system cancers. Only original full-text publications written in English have been analyzed. Studies involving treatment with quinolones (alone or in combination with standard chemotherapeutic agents) for normal and cancer cells other than those originating from the genitourinary system were also included. Additionally, the PubMed database was searched for information about concentrations achievable in urine and prostate tissue after oral or intravenous administration of different quinolones. The databases were reviewed until the end of June 2021.

Extracted data included the following elements: type of quinolones and their derivatives, quinolone concentrations, type of tested cells, incubation time, influence on cell viability, changes induced in cells, measurement results, the way of assessing results, and the effectiveness of tested compounds. Among the 17 quinolones described, antitumor activity was examined for 15 of them, and no cytotoxic effects of grepafloxacin and finafloxacin have been analyzed so far in the literature. For other compounds, 54 full-text articles were included for the analysis of anticancer properties of quinolones. Detailed data extraction for each quinolone is presented in Figure 2 . In order to search for information on quinolone concentrations achievable in prostate tissue or urine, combination of quinolone name and “prostate” or “urine” was used. Articles containing necessary information were chosen for Tables 1 , 2 preparation.

Most of the analyzed quinolones reach high concentrations in the urine ( Table 1 ). In the case of nalidixic acid and pipemidic acid, the antitumor properties against bladder and prostate tumor cell lines have not yet been tested. These compounds, although exhibiting promising properties, confirmed in leukemia, osteosarcoma, and ovarian, breast, and pancreas cancers, belong to the old generation and are currently less frequently used. Similar to the first generation of quinolones, lomefloxacin reaches high concentrations in the urine. However, due to phototoxicity and the lack of effective action of this drug on cancer cells, its use is limited. A solution to this problem may be the modification of lomefloxacin, which could increase its effectiveness against cancer cells and reduce its toxic effects. Moxifloxacin and pefloxacin have also not been yet tested against genitourinary tract cancer cells. The analyzed databases contain many publications about the cytotoxic properties of these chemotherapeutic agents against other cancer cells, like leukemia and colon, pancreas, and breast cancers. Considering the urinary accumulation at 130 μg/ml, this compound could potentially be used in the treatment of bladder cancer. Finafloxacin also reaches high concentrations in the urine; however, its activity has not been tested against cancer cells so far. Marginal concentrations in the urine are reached by compounds such as grepafloxacin, sparfloxacin, trovafloxacin, and gemifloxacin, which precludes them for use in the treatment of genitourinary system cancers ( Table 1 ).

Ciprofloxacin, ofloxacin, fleroxacin, enoxacin, norfloxacin, and levofloxacin were the only quinolones tested on bladder cancer cell lines (EJ, T24, J82, BOY, HTB9, HT1197, HT1376, TCCSUP, MBT-2, and BC-5867); however, the majority of the compounds mentioned above (except enoxacin and norfloxacin) were tested only on the T24 cell line ( Table 3 ). Most of the research was carried out using ciprofloxacin. IC₅₀ presented in Table 3 was, in most cases, due to lack of specific values, calculated on the basis of available data in analyzed manuscripts. Data from the study of Ebisuno et al. were not included in the analysis due to high differences in cell viability calculated with two different methods (29). Based on the collected data, it can be concluded that enoxacin has a comparable activity like ciprofloxacin and norfloxacin on the EJ cell line due to a similar IC₅₀ value after 72 h of incubation. Ciprofloxacin has a stronger activity than levofloxacin, ofloxacin, and fleroxacin due to the lower IC₅₀ values obtained for three (T24, J82, and TCCSUP) or one (T24) tested cell line, respectively. Levofloxacin has better cytotoxic properties against the T24 cell line and similar to the BOY cell line compared to ofloxacin. The calculated IC₅₀ values of ciprofloxacin, norfloxacin, enoxacin, and levofloxacin are achievable in the urine ( Table 1 ), which makes these compounds possible in the treatment of bladder cancer. The IC₅₀ values calculated for ofloxacin after the 400-mg dose in most cases ( Table 3 ) are not achievable in urine, limiting this compound’s use. In the case of fleroxacin, the IC₅₀ values obtained after 12 h of incubation with T24 and MBT-2 cell lines are not achievable in urine. The solution to this problem may be the use of a higher dose of those quinolones and their long-term use. Long-term therapy using these quinolones is safe and does not cause side effects. In addition, ciprofloxacin and ofloxacin (no data for levofloxacin) show a stronger cytotoxic effect in acidic pH, and such conditions are present in urine. The results discussed above indicate that all quinolones presented in Table 3 are promising candidates for use in the treatment of bladder cancer. According to collected data, ciprofloxacin seems to be the most effective due to the lowest IC₅₀ values calculated for all tested cell lines. Low IC₅₀ values were also calculated for norfloxacin, levofloxacin, and enoxacin, but compared to ciprofloxacin, a small number of data available in literature do not allow to draw reliable conclusions about their effectiveness. In our recent study, we additionally, for the first time, analyzed the influence of ciprofloxacin and levofloxacin on bladder cancer cells in 3D culture (spheroids). We showed that IC₅₀ values calculated after drug incubation with cells in standard 2D culture were not effective in 3D culture. Only the concentration reducing 90% of cell viability was effective in cell viability reduction and caspase 3/7 activation in 3D culture. These concentrations were 1,500 and 245 µg/ml for ciprofloxacin and 4,670 and 730 µg/ml for levofloxacin, respectively, after 24 and 48 h of incubation with drugs. The concentration obtained after 48 h of incubation with ciprofloxacin is achievable in urine, which suggests the potential of this drug in clinical application. Additionally, we tested both drugs on a non-malignant uroepithelial cell line. Our results showed that both tested drugs were more effective against cancer cell lines which were more pronounced in 3D culture (62).

There is a lack of data in the literature about the concentration of some quinolones in prostate tissue. This concentration has not been determined for nalidixic and pipemidic acids, grepafloxacin, sparfloxacin, gemifloxacin, and finafloxacin. The use of other quinolones, in most cases, allows reaching a higher concentration in the prostate tissue than in the serum ( Table 2 ). The highest concentration was obtained after the use of levofloxacin; high concentrations were also obtained for lomefloxacin and moxifloxacin. In the case of other quinolones, good penetration of the drug into the prostate gland was observed, which allowed achieving over two times higher concentrations in this organ than in the serum. In comparison to the concentrations obtained in urine, the concentrations obtained in the prostate gland are much lower, which limits the use of quinolones alone in the treatment of prostate cancer. In the case of gatifloxacin, the prostate tissue dose was not determined; however, its concentration in the prostatic secretion, seminal fluid, and ejaculate was tested, reaching the highest concentration at 3.10 μg/ml.

Only five quinolones (ciprofloxacin, levofloxacin, enoxacin, norfloxacin, and gatifloxacin) have been so far studied on prostate cancer cell lines (LNCaP, 22Rv1, VCaP, PC3, and DU-145). In six studies, the use of ciprofloxacin was especially focused on prostate cancer, and the other two studies analyzed modified quinolones on the panel of various cell lines; therefore, information about prostate cancer is very limited ( Table 3 ). The PC3 cell line was analyzed in almost all studies. In the study of El-Rayes et al. and Sousa et al., only one concentration of ciprofloxacin was tested, which is why it is not possible to calculate IC₅₀ values (75, 76). In another study, values presented on the graph do not allow for IC₅₀ calculation; only information showing that ciprofloxacin is less toxic for normal prostate epithelium (MLC88991) can be obtained (77). Ninety percent of inhibition of prostate cancer cell growth was observed at a concentration of 50 μg/ml; this concentration is difficult to obtain in the prostate gland ( Table 2 ). After application of enoxacin at a concentration of 40 μg/ml for 5 days, inhibition of cell growth of all five prostate cancer lines was observed in the range of 17%–59%; the most sensitive cell line was LNCaP. In the study of Pinto et al., the IC₅₀ values for ciprofloxacin were 254 and 172 μg/ml for PC3 and LNCap, respectively; after a 24-h exposure, this value decreased to 70 μg/ml for both cell lines after a 72-h exposition to this drug (71, 72). In another study, the IC₅₀ value calculated for this same cell line after the 24-h exposition was below 16 μg/ml, which is a significantly lower dose than that calculated by Pinto et al. (73). Levofloxacin, compared to ciprofloxacin, was more effective against the DU-145 cell line after a 24-h incubation, while after 48 h the situation was opposite. In this study, the advantage of both drugs over cancer cells was demonstrated (62). In the case of norfloxacin, it was shown that prostate cancer cells were the most resistant to this drug among all tested cancer cell lines. The IC₅₀ value calculated for norfloxacin was approximately 11.2 μg/ml, similarly in the case of ciprofloxacin; these concentrations are over two times higher than the maximum values obtained in prostate tissue (74). In the case of gatifloxacin, only its modified forms were examined; the experiment was performed on a panel of 58 cancer cell lines, including two prostate cancer cell lines (PC3 and DU145). Etoposide was used as a control (78). The IC₅₀ value obtained for prostate cancer was approximately 6 μg/ml, which is a relatively low concentration. However, it is almost twice as high as that obtained in prostatic secretion. Moreover, a modified compound of gatifloxacin was less effective than that of etoposide. Although quinolones accumulate at a higher concentration in prostate tissue than in serum, reachable concentrations are much lower than these IC₅₀ values calculated for ciprofloxacin, norfloxacin, and modified gatifloxacin. This problem may be resolved by the use of modified quinolones like nanocomposites. The use of such compounds significantly improves anticancer activity by reducing the toxic dose; however, it is not known whether the modification of quinolones will affect its accumulation in prostate tissue. Another solution is the improvement of quinolone accumulation in prostate tissue using direct injection to prostate tissue or use modification of drug delivery systems like lipid-based complexes. The obtained results indicate that quinolones cannot be used as independent anticancer drugs in the treatment of prostate cancer; the solution may be their use in combination therapy with standard-use chemotherapeutics.

The mechanism of action of quinolones is mainly based on the inhibition of bacterial topoisomerase II; however, they also show activity against analogous enzymes in eukaryotic cells. Other changes in eukaryotic cells associated with quinolones’ mechanism of action are shown in Supplementary Table 1 . One of the mechanisms of action is the dysfunction of the mitochondria, which, according to Lawrence et al., can be linked with the theory of endosymbiosis and similarity in the structure of mitochondria to bacterial cells to which quinolones are active (79). The addition of quinolones to cell culture led to cell cycle arrest, mainly in the S and G2/M phases. In the case of moxifloxacin, levofloxacin, and sparfloxacin complexes with gold (III), a higher number of cells in the G0/G1 and subG1 phases were observed compared to control. Inhibition of topoisomerase led to the inhibition of DNA synthesis and its fragmentation. Downregulation of TOP2A and TOP2B genes, two isomers of topoisomerase II, were observed after ciprofloxacin and levofloxacin treatment (62). Quinolones induced cell death mainly through apoptosis, rarely necrosis (62, 75, 77, 80–82). Apoptosis was not observed after the use of gatifloxacin and moxifloxacin. Also, studies of the level of apoptosis indicators such as caspase-3, Bax, and Bcl-2 protein or ROS were carried out. Bax and Bcl-2 are intracellular proteins that are known to regulate apoptosis. The Bax protein inactivates the Bcl-2 protein that protects cells against apoptosis, thereby increasing the Bax/Bcl-2 ratio. An increase in the level of this factor may be responsible for the induction of apoptosis. In addition, Bax translocation into the mitochondria is thought to accelerate the onset of apoptosis. Mitochondrial membrane permeability disorders were also observed, which led to the activation of caspases and DNA fragmentation (77). Caspase-3 is an important enzyme that is required for the final phase of apoptosis. ROS has also been shown to stimulate apoptotic pathway signaling by activation of caspase-3 (83). The effect of quinolones on metastasis treatment was also analyzed; gatifloxacin showed a reduction in the migration and invasion of cancer cells by influencing epithelial to mesenchymal transition (EMT) (84, 85). Also, changes in TP53 and CDKN1 gene expression after treatment with ciprofloxacin or levofloxacin were observed (62, 86). Analysis of differentially expressed genes (DEGs) using the STRING database version 11.5 allows for detection of three pathways, which could play a potential role in fluoroquinolones’ action. The highest strength (above 1.9) was noticed for apoptosis, platinum drug resistance, and p53 signaling pathway. Based on analyzed publications, the potential mechanism of quinolones’ action was summarized as shown in Figure 3 . Many studies indicate that quinolones in combination with other commonly used anticancer drugs (etoposide, cisplatin, doxorubicin, epirubicin, imatinib, 5-fluorouracil, irinotecan, docetaxel, camptothecin, tamoxifen, etc.) and as complexes with metals such as copper, platinum, ruthenium, zinc, and gold have better cytotoxic effects (5). Metallic derivatives of antibacterial drugs are gaining more and more interest because the coordination of metal by a synergistic effect leads to various pharmacological activities, including antiproliferative, antimicrobial, antifungal, and antiviral activities (87). Quinolones like etoposide, doxorubicin, and mitoxantrone have the same cellular target—topoisomerase II—but distinct mode and sites of action within this enzyme (72). Etoposide works by suppressing the ability of topoisomerase II to ligate DNA molecules, whereas quinolones have little effect on ligation but stimulate the rate of DNA cleavage by topoisomerase II (88). Additionally, etoposides’ action leads to the release of pro-inflammatory cytokines, such as IL-8, TNF-α, and IL-1β, which, depending on the tumor cell line, is inhibited by quinolones. The release of proangiogenic IL-8 is undesirable and considered as a side effect of the use of etoposide (89). Doxorubicin, similar to ciprofloxacin, helps to stabilize double-stranded DNA complexes with topoisomerase II; the difference is that ciprofloxacin is a non-intercalating drug (71). In such cases, when the mechanism of action is similar, the treatment with two drugs should be sequential, not simultaneous (71). Different mechanisms of action show drugs like cisplatin, imatinib, vinblastine, 5-fluorouracil (5-FU), and docetaxel. Cisplatin poses the ability to bind with purine bases and interfere with DNA repair mechanisms, causing damage to genetic material, and the consequent induction of apoptosis enhances the activity of quinolones (90). Vinblastine and docetaxel interact with mitotic spindle, imatinib prevents platelet-derived growth factor receptor activation, and 5-FU is classified as a pyrimidine anti-metabolite leading to inhibition of DNA synthesis (64, 71, 72). Although these drugs have different metabolic pathways and generally act synergistically, they can affect the same molecular process like G2/M arrest or apoptosis induction by Bcl-2 downregulation. That is why in order to obtain the best therapeutic effect, it is important to choose the right drug combination and method of its administration (71, 72). Not all studies compared the properties of quinolones against cancer and normal cells; however, if such analyses were performed, quinolones did not affect or showed little activity on normal cells, which means that they show selective action against cancer cells (74, 77, 91–96). Modification of quinolones’ structure can strengthen their properties (97–105). However, in such cases introduction of a new drug on the market is difficult due to expensive and long-term procedures including all stages of clinical trial conduction. The advantage of unmodified drugs is the possibility of their repositioning without such complicated procedures. Quinolones can also be used in combined therapy as a supportive drug, which can lead to improvement in treatment efficacy (106–110).

It should be also mentioned that, beside direct action, through topoisomerase II inhibition, quinolones can also affect the neoplastic process through indirect effects, by modulation of the immune response (111). The immunological system plays an essential role in cancer development and progression, and it can detect and destroy abnormal cells preventing or slowing down cancer growth. However, cancer cells develop different mechanisms, which allows avoiding their destruction by the immune system (112). Enhancing the immune response can lead to the stronger elimination of cancer cells. Fluoroquinolones can induce an immunomodulatory effect by activation or inhibition of specific cytokines. It was shown that fluoroquinolones, like ciprofloxacin, are able to upregulate interleukin 2 (IL-2) production, which is the most important growth factor for lymphocyte T and NK cells. This effect, together with activation of IL-3 and GM-CSF synthesis, which stimulates bone marrow generation, could be significant in immune-compromised cancer patients (113). Additionally, fluoroquinolones inhibit pro-inflammatory cytokines, like IL-1β, IL-6, IL-8, and TNF-α, which together with stimulation of IL-2 can enhance immunomodulatory effects. Inhibition of IL-8 by moxifloxacin was observed in colon cancer and IL-1β and TNF-α in leukemia cells (88, 89, 109). IL-1β and IL-8 are involved in the NFκβ signaling pathway leading to cell survival, and their downregulation could have an impact on cell death induction. The TNF signaling pathway can activate antiapoptotic and pro-survival signals and can lead to apoptosis induction. Its downregulation can indicate that apoptosis of cancer cells could be induced not directly but indirectly by the inhibition of pro-survival signals ( Figure 3 ). On the other hand, fluoroquinolones can increase the level of anti-inflammatory cytokines, like IL-10, which inhibit the production of IL-2, IL-3, or GM-CFS (111). That is why, in order to learn more about the mechanism of action of quinolones on regulation of the immune system in cancer cells, more experiments have to be performed.