PRCC) is the most common nonclear cell RCCs and is known to comprise two histological subtypes. PRCC2 is more aggressive and is molecularly distinct from the other subtypes. Despite this, PRCCs are treated together as one entity, and they show poor response to the current therapies that do not target pathways implicated in their pathogenesis. We have previously detected ABCC2 (an ABC transporter), VEGF, and mTOR pathways to be enriched in PRCC2. In this study, we assess the therapeutic potential of targeting these pathways in PRCC2. Twenty RCC cell lines from the Cancer Cell Encyclopedia were compared to the Cancer Genome Atlas PRCC cohort (290), to identify representative PRCC2 cell lines. Cell lines were further validated in xenograft models. Selected cell lines were treated in vitro and in vivo (mice models) under five different conditions, untreated, anti‐VEGF (sunitinib), ABCC2 blocker (MK571), mTOR inhibitor (everolimus) and sunitinib + MK571. Sunitinib +ABCC2 blocker group showed a significant response to therapy compared to the other treatment groups both in vitro (P ≤ 0.0001) and in vivo (P = 0.0132). ABCC2 blockage resulted in higher sunitinib uptake, both in vitro (P = 0.0016) and in vivo (P = 0.0031). Everolimus group demonstrated the second best response in vivo. The double‐treatment group showed the highest apoptotic rate and lowest proliferation rate. There is an urgent need for individualized therapies of RCC subtypes that take into account their specific biology. Our results demonstrate that combined targeted therapy with sunitinib and ABCC2 blocker in PRCC2 has therapeutic potential. The results are likewise potentially significant for other ABCC2 high tumors. However, the results are preliminary and clinical trials are needed to confirm these effects in PRCC2 patients.Papillary renal cell carcinoma ( ABC transportersATP binding cassetteABCC2ABC binding cassette subfamily C member 2CCRCCclear cell renal cell carcinomaChrchromosomePRCCpapillary renal cell carcinomaet al., et al., Renal cell carcinomas (RCCs) are tumors that arise from renal tubules. They represent ~90% of adult kidney tumors and one of the most prevalent malignancies worldwide  (~75%), and the second most common are papillary RCC (PRCC) (10–15%) which is comprised of multiple subtypes  tyrosine kinase inhibitors (TKIs), for example, sunitinib and pazopanib, and second‐line therapies the mammalian target of rapamycin (mTOR) inhibitors (everolimus and temsirolimus), and the recently approved PD‐1 immune checkpoint inhibitor nivolumab , with high statistical significance , and CCLE (Cancer Cell Line Encyclopedia‐Broad Institute). Gene expressions  were downloaded for the KIRP 290 PRCC cohort from the TCGA Web site (our published study)  mice (4 mice). Tumors were harvested and sections were histologically examined for cell lines morphology and IHC profiles and compared to those of PRCC1. Similar to PRCC1, the histology of the mice tumors showed a tubulo‐papillary architecture of small low‐grade tumor cells, linear nuclear arrangement, and negative staining for ABCC2 IHC  as a PRCC1 cell line based on literature evidence of its papillary nature  and the Deutsche Sammlung von Mikroorganismen und Zellkulturen , respectively. The former was grown in McCoy cell media with 10% fetal bovine serum (FBS), while the latter was grown in DMEM cell media with 15% FBS, 0.4 μg·mL3 cells per well in a 96‐well plate and after 24 h grouped into the five treatment conditions, untreated, treated sunitinib 1.0 μm, treated with MK‐571 25 μm (ABCC2 blocker) . The plate was scanned with image express analyzer to detect the number of cells with dye uptake. Subsequently, the cells were fixed with paraformaldehyde (PFA), subjected to the Hoechst dye again, and rescanned with the image express.nu) mice and NOD SCID (severe combined immunodeficiency) gamma mice.Mice bought from ‘The Jackson laboratory’ were the immunocompromised strains outbred athymic nude 2 x π /6, where length is the longest dimension and diameter is the shortest dimension , sunitinib only (50 mg·kg−1), everolimus only (2.5 mg·kg−1), sunitinib + MK571, and everolimus + MK571.CAKI‐2 cells were grown to 80% confluency and trypsinized, into single‐cell suspension. 10et al., Immunohistochemistry was performed using Ki67 and ABCC2 antibodies. The Ki67 was performed using a Ventana automated system. Ki67 is a marker of cell proliferation (detected by nuclear staining), as it is present in all active phases of the cell cycle .Mice tumor slides stained with Ki67 were scanned and analyzed with the et al., et al., graphpad prism 7 and spss statistical software .From the PRCC TCGA cohort, a distinct PRCC2 mRNA signature of 500 genes (compared to PRCC1) was elicited , as well as best correlation with the ARE pathway genes  Figs Of the 19 RCC cell lines, CAKI‐2 showed excellent correlation with PRCC2 top matched genes  mice (6 mice). Tumors were harvested and sections were histologically examined for cell lines morphology and IHC profiles and compared to those of PRCC2. All of the six injected nude mice developed tumors with papillary morphology and had large eosinophilic cells, with nuclear pseudostratification and prominent nucleoli, consistent with what is known as a PRCC2 morphology  ± MK571. The group of cells that were exposed to the dual treatment with both sunitinib and blocker was the most sensitive to treatment  and everolimus did not produce significant differences in cell viability versus the blocker alone  treated groups were stained with the cell permeable Hoescht 33342 DNA dye and then subjected to image screening and visualization using the Xpress image analyzer. MK571‐treated cells showed considerable dye uptake (indicating dye retention), whereas untreated cells failed to stain indicating an active export of the Hoescht dye by ABCC2. Then, we proceeded to fix the cells with paraformaldehyde (PFA) to terminate the ABCC2 drug transporter pump effect. Fixed untreated cells showed a significant increase in staining after blocking the ABCC2 effect with fixation, et al., To further confirm that ABCC2 contributes to drug resistance through pumping out medications, we measured sunitinib uptake in CAKI‐2 cells through the detection of its known spectral range in flow cytometry  was observed in the group that was exposed to the sunitinib and MK571 treatment  and 3686 mm3 in the control untreated group (P = 0.0132). The growth curves of everolimus‐treated group showed the second highest response to therapy, volume of 1225 mm3. Both everolimus and sunitinib + MK571 arms showed significant differences from the sunitinib treated arm. Sunitinib only and MK571 only treated groups showed similar responses to their corresponding therapy  with no significant differences between the two treatment arms.Tumor mouse model was optimized in immunocompromised athymic nude and NOD SCID gamma mice. 10P = 0.0045) compared to other treatment groups  , which was significantly higher , followed by sunitinib (14%), everolimus (7.5%) and then MK571 and untreated groups (2–5%). Upon comparing the distant organ metastasis among the different groups, the sunitinib + MK571 and everolimus groups showed the lowest level of metastasis among the treated mice (25–30%) (number of mice showing distant organ metastasis in their group). Even though the everolimus group showed a low level of lung metastasis, it was less tolerated by mice as 50% (2/4) were deceased halfway through the treatment period. The sunitinib and MK571 groups showed 50% and 75% metastasis, respectively Table .in vivo analysis yielded similar results to what was detected with in vitro treatment, with a significant increase in intracellular sunitinib among the group that was additionally treated with the ABCC2 blocker. sunitinib median fluorescent intensity (MFI) was 11 250 in the sunitinib + MK571 treated mice versus 6870 in the sunitinib only treated mice (P = 0.028) . After tumor dissociation into a single‐cell suspension, the sunitinib content of each mouse tumor was measured by flow cytometry as described in previous sections. Our et al., et al., et al., et al., et al., In the era of personalized medicine, we are getting to understand more about the individualized biology of different tumors and our approach to this management should reflect this knowledge. While there are many drugs under development toward that goal, we are not yet there for the different RCC subtypes  in PRCC2  and enrichment in the mTOR pathway, consistent with TCGA findings  did not show an increase in sunitinib uptake upon ABCC2 blockage. The results ascertain what was shown in other studies regarding an increase in drug uptake upon ABC transporter blockage, especially in cancer with a high level of transporters  have attempted to compare between sunitinib and everolimus treatment in PRCCs . These data highlight potential benefit in subtyping PRCCs in clinical trials. Large subtype‐specific clinical trials are required to formulate better guidelines  RCC cell line. in vitro setting and in mouse models. However, our findings would pave the way for clinical trials that can confirm these results and lead to a more accurate tumor‐specific treatment for the patients of PRCC2 and similar tumors.We believe our study uncovers extremely pertinent information regarding PRCC2 treatment. Discoveries on the individualized nature of tumor biology should direct the clinical field toward similarly individualized treatments. We assessed the efficacy of targeting tumor‐specific pathways in a preclinical setting and showed that these treatment modalities show great promise. We highlight the importance of targeting ABC transporters, as ABCC2 in PRCC2 and in other high drug transporter tumors. We acknowledge that the current study has a number of limitations, particularly its experimental design in cell lines and the nature of the assessment in in vivo and in vitro experiments and analyzed data. ZL guided and performed experiments, and interpreted results. FB reviewed and helped interpret the tumor pathology data. JB helped with the in vivo experiments. GB reviewed data and guided the tumor therapeutic experiments, and helped interpret data. AF reviewed and helped interpret the clinical data. FB performed experiments and immunohistochemical stains. MD: reviewed and helped interpret the tumor pathology data. GS, senior scientist, supervised the project, guided the experimental framework, helped interpret the results, and reviewed the final work and the manuscript.RS designed the project, performed the experiments, analyzed the data, and wrote the manuscript. MF performed Fig. S1. The CAL‐54 RCC cell line represents PRCC1.Fig. S2. The CAKI‐2 cell line represents PRCC2.Table S1. Histological examination of mice tumor models.Click here for additional data file.


Additionally, analogues of perillyl alcohol, a monoterpene with known anticancer activity, were evaluated to identify the molecular characteristics which contribute to their cytotoxicity, which was tested against OVCAR-8, HCT-116, and SF-295 human tumor cell lines, using the MTT assay. The results of this study showed that (−)-perillaldehyde 8,9-epoxide exhibited the highest percentage inhibition of cell proliferation (GI = 96.32%–99.89%). Perillyl alcohol exhibited high cytotoxic activity (90.92%–95.82%), while (+)-limonene 1,2-epoxide (GI = 58.48%–93.10%), (−)-perillaldehyde (GI = 59.28%–83.03%), and (−)-8-hydroxycarvotanacetone (GI = 61.59%–94.01%) showed intermediate activity. All of the compounds tested were less cytotoxic than perillyl alcohol, except (−)-perillaldehyde 8,9-epoxide (IC50 = 1.75–1.03 µL/mg). In general, replacement of C-C double bonds by epoxide groups in addition to the aldehyde group increases cytotoxicity. Furthermore, stereochemistry seems to play an important role in cytotoxicity. We have demonstrated the cytotoxic influence of chemical substituents on the p-menthane structure, and analogues of perillyl alcohol.Compounds isolated from essential oils play an important role in the prevention and treatment of cancer. Monoterpenes are natural products, and the principal constituents of many essential oils. The aim of this study was to investigate the cytotoxic potential of  Cancer is a disease in which certain cells in the body grow in an uncontrolled way. It is one of the world’s most serious illnesses, and together with heart attacks, it kills more people than any other disease. Its hallmarks constitute an organizing principle for rationalizing the complexities of neoplastic diseases. They include sustained proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis .Nature is an important source of new candidates for therapeutic compounds; a large chemical diversity is found in several species of plants, animals, and microorganisms . PrakashVepris macrophylla and Myristica fragrans in vitro  [14 [15  exhibit rocesses ,35,36.50 of compound 16, besides presenting the highest percentage of inhibition of cell proliferation, 16 was more potent than 3, showing high cytotoxicity in all three cell lines, with IC50 values ranging from 1.03–1.75 µg/mL, as shown in Evaluating the IC16 showed high cytotoxicity in tumor cells, it was tested for its ability to induce lysis in mouse erythrocytes (data not shown). The mechanical stability of the erythrocyte membrane is a good indicator of in vitro damage in cytotoxicity assays, since many drugs can alter this delicate structure [16 was not hemolytic even at the highest tested concentration (500 μg/mL). This suggests that the cytotoxicity mechanism is probably related to a more specific pathway.Since compound tructure . Red blotructure ,39,40,4116, ½ IC50 (0.32 µg/mL), IC50 (0.64 µg/mL) and 2 × IC50 (1.28 µg/mL) were chosen against HL-60. The anti-proliferative effects of 16 were demonstrated through the trypan blue dye exclusion test (Three concentrations of compound ion test . It show16), in cancer cells, two tests were performed, fluorescence microscopy using acridine orange/ethidium, and hematoxylin-eosin coloration analyzed by optical microscopy. After 24-h incubation, the effects of 16 were evaluated based on cell morphology using hematoxylin-eosin. All concentrations and showed severe drug-mediated changes. The hematoxylin-eosin stained HL-60 cells treated with 16 presented a morphology consistent with apoptosis, including a reduction in cell volume, chromatin condensation, and nuclei fragmentation, as well as, necrosis with membrane disruption, cell swelling and rupture leading to inflammation. The acridine orange/ethidium bromide stained and treated cells also displayed a morphology consistent with apoptosis and necrosis, in a concentration-dependent manner (p < 0.05) (Attempting to ascertain the cellular death process (as induced by  < 0.05) .16 caused cell death by apoptosis in the three concentrations tested, as shown in Compound 16 presented necrotic cell death simultaneously. This type of cell death is characterized by swelling of cellular organelles, cell membrane rupture, cell lysis (unlike apoptosis), and the core becomes distended yet substantially intact. Necrotic death is typically followed by inflammatory reactions [16 is increased.Besides the apoptotic process in doses of 0.64 and 1.28, compound eactions . This ap16 as a possible candidate for in vivo tests, even with the two processes of cell death responsible for its in vitro cytotoxicity.Various chemical agents such as β-lapachone described by Li and collaborators , induce This evaluation is important due to the fact that perillyl alcohol and its analogues are structurally similar to many of the chemical constituents of essential oils and other natural products. Therefore the results obtained in this study may well provide a reference for the development of novel cytotoxic agents having an appropriate biological profile.15) displayed good cytotoxic activity, with a GI value of 59.28%–83.03%. Compounds 14 , and 15  were less cytotoxic  than 16 . This indicates that the presence of an epoxide and/or aldehyde function in the molecule is important for cytotoxicity. Further, the anti-proliferative activity was even more pronounced when these functional groups  are both in the chemical structure, since (+)-limonene 1,2-epoxide (12) (GI = 58.48%–93.10%), which contains only the epoxide group, was less bioactive than 16 (GI = 96.32%–99.89%). On the other hand, comparing (+)-limonene 1,2-epoxide (12) with (−)-carvone epoxide (5), it appears that the endocyclic ketone function does not contribute to cytotoxicity, as compound 12 (GI = 58.48%–93.10%) was approximately 2.5- to 11-fold more cytotoxic than 5 (GI = 8.21%–29.24%).The presence of an exocyclic aldehyde carbonyl and the conjugated C-C double bond seem to contribute to cytotoxicity, as (−)-perillaldehyde (14) and (−)-perillaldehyde (15) differ from each other only by a functional group. Compound 14 has a hydroxyl group whereas 15 presents an aldehyde function, both located at the same position in the p-menthane skeleton (C-7). Perillyl alcohol (14) (GI = 90.92%–95.82%) exhibited higher cytotoxicity compared to (−)-perillaldehyde (15) (GI = 59.28%–83.03%), suggesting that a hydroxyl group at C-7 position produces in a more potent inhibition of cell proliferation than an aldehyde group at the same position.The monoterpenes perillyl alcohol (1) (GI = 2.28%–12.28%), (+)-carvone (2) (GI = 34.39%–48.07%), (+)-pulegone (9) (GI = 10.25%–27.44%), and (−)-trans-isopulegone (11) (GI = 5.98%–18.96%). In the general, the evaluated ketones showed moderate to weak cytotoxicity, but considerable differences in their potencies were observed. We also noted that compounds 9  and 11 (saturated ketone) displayed different cytotoxic effects against the three tumor cell lines.To investigate the effect of the disposition of the ketone carbonyl group in cytotoxic activity, we compared the oxygenated monoterpenes (−)-carvone (1) (GI = 2.28%–12.28%) with (−)-carvone epoxide (5) (GI = 8.21%–29.24%) and (+)-pulegone (9) (GI = 10.25%–27.44%) with (R)-pulegone oxide (10) (GI = 16.02%–43.21%). Similar results were found comparing the compounds (+)-carveol epoxide (6) and (−)-cis-carveol (7), and (−)-perillaldehyde (15) and (−)-perillaldehyde 8,9-epoxide (16). Replacement of a non-conjugated C-C double bond by an epoxide function in 7 (GI = 3.61%–21.16%) and 15 (GI = 59.28%–83.03%), resulting in the respective compounds 6 (GI = 4.58%–35.35%) and 16 (GI = 96.32%–99.89%), produces a subtle increase in cytotoxicity.Replacement of a carbon-carbon double bond, conjugating the carbonyl with an epoxide group resulted in a modest enhancement of anti-proliferative effect. Improvement of the biological potency was observed thru comparison of (−)-carvone (5) with (R)-pulegone oxide (10). Compounds 5 and 10 present a ketone and epoxide group in different positions in the p-menthane skeleton, and different biological effects for these monoterpenes was observed. (R)-Pulegone oxide (10) was more bioactive (GI = 16.02%–43.21%) than (−)-carvone epoxide (5) (GI = 8.21%–29.24%), suggesting that the position of these groups in the p-menthanic structure influence the cytotoxic activity.To ascertain the influence of the disposition of the epoxide and ketone groups in cytotoxicity, we compared (−)-carvone epoxide (1) was compared with (−)-8-hydroxycarvotanacetone (3), and (+)-carvone (2) with (+)-8-hydroxycarvotanacetone (4). Compound 1 (GI = 2.28%–12.28%) was significantly less cytotoxic than 3 (GI = 61.59% to 94.01%), indicating that the presence of a hydroxyl group causes an enhancement of cytotoxicity. However, (+)-carvone (2) (GI = 34.39%–48.07%) was more bioactive than its corresponding monoterpene alcohol (+)-8-hydroxycarvotanacetone (4) (GI = 3.12%–16.36%). In addition, we also evaluated the effect of adding a second hydroxyl group to the p-menthane structure. It was observed that (−)-sobrerol (13) (two hydroxyl groups) showed a significant decrease in the inhibition rate of cell proliferation as compared to (−)-8-hydroxycarvotanacetone (3) (one hydroxyl group), with GI values of 9.78%–41.10% and 61.59%–94.01%, respectively. Thus, these results revealed that the more polar monoterpenes, such as (−)-sobrerol (13), are less effective inhibitors of cellular proliferation.To examine whether the introduction of a hydroxyl group (hydroxylation of a double bond) in the molecular structure affects the cytotoxic activity, (−)-carvone (3) with (−)-acetoxycarvotanacetone (8), and (S)-(−)-perillyl alcohol (14) with both (−)-perillyl acetate (17), and (S)-perillyl benzoate (18). (−)-8-hydroxycarvotanacetone (3) (GI = 61.59%–94.01%) presented more potent cytotoxic activity than its acetate derivative, (−)-8-acetoxycarvotanacetone (8) (GI = 1.62%–30.47%), suggesting that the replacement of a hydroxyl group by an acetate causes a marked decrease in the cytotoxicity. Additionally, compound 14 was more cytotoxic (GI = 90.92%–95.82%) than 17 (GI = 3.06%–16.41%) and 18 (GI = 2.86%–5.02%), probably due to its lower lipophilicity. (S)-Perillyl benzoate (18), the most lipophilic compound, was found to be the least potent inhibitor of cellular proliferation for all of the cell lines evaluated.To investigate whether the presence of an ester function in the chemical structure alters the cytotoxicity, we compared (−)-8-hydroxycarvotanacetone (1) and (+)-carvone (2), and (−)-8-hydroxycarvotanacetone (3) and (+)-8-hydroxy- carvotanacetone (4). (+)-Carvone (2) (GI = 34.39%–48.07%) was more cytotoxic than its enantiomer (−)-carvone (1) (GI = 2.28%–12.28%) in all the cancer lines tested. Similarly, (−)-8-hydroxy- carvotanacetone (3) exhibited better cytotoxic effect than its enantiomeric form (+)-8-hydroxy- carvotanacetone (4), with respective GI values of 61.59%–94.01% and 3.12%–13.36%. The findings suggest an association between the cytotoxicity of the monoterpenes tested and their stereochemistry.To evaluate the influence of chirality on cytotoxicity, we compared the pairs of enantiomers (−)-carvone (R)-pulegone oxide [cis-carveol [trans-isopulegone [S)-perillyl benzoate [1H- and 13C-NMR. (+)-Pulegone, (−)- and (+)-carvone, and (S)-(−)-perillyl alcohol were purchased from Sigma-Aldrich Co. . The chemical structures of the evaluated compounds are shown in The compounds -carne oxide , (−)-cis-carveol , (+)-car-carveol , (+)-lim-carveol , (−)-per-carveol , (−)-per-carveol , (−)-per-carveol , (−)-trapulegone , (+)- anpulegone , (−)-8-apulegone , (S)-perbenzoate , and (−)benzoate  were pre2 atmosphere.The cytotoxicity of the 18 compounds was tested against OVCAR-8 (ovarian adenocarcinoma), HCT-116 (colon carcinoma), SF-295 (glioblastoma), and HL-60 (leukemia) human cancer cell lines, all obtained from the National Cancer Institute, Bethesda, MD, USA. Cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 µg/mL streptomycin, and 100 U/mL penicillin, and incubated at 37 °C in a 5% COH-tetrazolium bromide  to a purple formazan product, as described by Mossman [6 cells/mL in 100 μL medium). After 24 h, the 18 compounds (25 μg/mL), were dissolved in 0.7% dimethyl sulfoxide (DMSO), and were added to each well . Then, the cells were incubated for 72 h at 37 °C in a 5% CO2 atmosphere. The experiment was performed as three independent experiments, using DMSO at 1%, and doxorubicin at 100 μg/mL as negative and positive controls, respectively. Doxorubicin, purity >98%; Sigma Chemical Co.).Tumor cell growth was determined by the ability of living cells to reduce the yellow dye 3--2,5-diphenyl-2 Mossman . For allAt the end of incubation, the plates were centrifuged, and the medium was replaced by fresh medium (150 μL) containing 0.5 mg/mL MTT. Three hours later, the formazan product was dissolved in 150 μL DMSO, and absorbance was measured using a multiplate reader . The treatment effects were expressed as the percentage of control absorbance of reduced dye at 595 nm. All absorbance values were converted into a cell growth inhibition percentage (GI%) by the following formula:50). The same protocol for the same cells was used to determine the IC50, varying only the concentration of the compound tested from 0 to 25 μg/mL, to verify the most potent compound. In addition, the IC50 was determined against HL-60 only for the most cytotoxic compound 16. Compounds with an IC50 value lower than 4 μg/mL were considered promising for the search for new anticancer agents [The compounds that caused greater degree of cell growth inhibition and which have not been described in the literature for cytotoxic activity against tumor cell lines were evaluated for their median inhibitory concentration able to induce 50% of maximal effect  were added to the suspension of red blood cells obtained from mice according to methodology adapted from Kang and collaborators [16 erythrocyte mixtures were incubated on a mixer for 60 min and then centrifuged at 3000 rpm for 5 min. Mixtures were incubated on a mixer for 60 min and then centrifuged at 3000 rpm for 5 min. After incubation at room temperature for 30 min and centrifugation, the supernatant was removed and the liberated hemoglobin was measured spectrophotometrically as the absorbance at 540 nm.The test was performed in 96-well plates using a 2% mouse erythrocyte suspension in 0.85% NaCl containing 10 mm CaClborators . Variousborators , Pita anborators , and Bezborators . The tub6 cells/mL) in absence or presence of different concentrations of compound 16 . After the incubation period of 24 h, 90 µL were removed from the cell suspension and added to 10 µL of trypan blue. Viable and non-viable cells were differentiated and counted in a Neubauer chamber [The cell viability was determined by the trypan blue dye exclusion test. HL-60 cells were seeded  was added, and the cells were observed under a fluorescence microscope. Three hundred cells were counted per sample and classified as viable, apoptotic or necrotic .6 cells/mL) in the absence or presence of different concentrations of compound 16 . After an incubation period of 24 h, 50 µL of cell suspension were transferred to cytospin slides, fixed with metanol for 60 s, and stained with hematoxylin-eosin [For morphological cell analysis, we used hematoxylin-eosin and examined using light microscopy. HL-60 cells were seeded  were obtained by nonlinear regression. Differences among the experimental groups were compared by one-way variance analysis (ANOVA), followed by Newman-Keuls test (p < 0.05). All analyses were carried out using the Graphpad program .Data are presented as mean ± SEM (or SD) or IC16 was the p-menthane derivative with the highest cytotoxic activity against the cancer cells lines tested. The process induced apoptotic and necrotic cell death. In addition the analysis of structure-activity relationships demonstrated that the greater anti-proliferative activity of 16 was determined by chemical aspects, such as presence of functional groups and their positions on p-menthane skeleton, suggesting that using appropriate structural modifications in these compounds, it may be possible to develop new cytotoxic agents. Therefore, compound 16 is a potential anticancer drug, yet requiring further tests to determine its in vivo antitumor activity.Based on the data of this study, we can conclude that among the 18 derivative compounds of perillyl alcohol tested, compound


Clonal VDJ rearrangement of B/T cell receptors (B/TCRs) occurring during B/T lymphocyte development has been used as a marker to track the clonality of B/T cell populations.We systematically profiled the B/T cell receptor repertoire of 936 cancer cell lines across a variety of cancer types as well as 462 Epstein-Barr Virus (EBV) transformed normal B lymphocyte lines using RNA sequencing data.Rearranged B/TCRs were readily detected in cell lines derived from lymphocytes, and subclonality or potential biclonality were found in a number of blood cancer cell lines. Clonal BCR/TCR rearrangements were detected in several blast phase CML lines and unexpectedly, one gastric cancer cell line (KE-97), reflecting a lymphoid origin of these cells. Notably, clonality was highly prevalent in EBV transformed B lymphocytes, suggesting either transformation only occurred in a few B cells or those with a growth advantage dominated the transformed population through clonal evolution.Our analysis reveals the complexity and heterogeneity of the BCR/TCR rearrangement repertoire and provides a unique insight into the clonality of lymphocyte derived cell lines.The online version of this article (10.1186/s12885-018-4840-5) contains supplementary material, which is available to authorized users. Clonal V(D)J  rearrangement which occurs during development of B/T lymphocytes has been used as a marker to track the clonality of B/T cell populations , 2. ThisIn this study, we systematically profiled the B/T cell receptor repertoire of 936 cancer cell lines across a variety of cancer types, as well as 462 Epstein-Barr Virus (EBV) transformed normal B lymphocyte lines, using RNA sequencing data from the Cancer Cell Line Encyclopedia (CCLE) . This stTranscriptome sequencing data were downloaded from the CCLE and Geuvadis RNA sequencing databases; and the B/T cell receptor repertoire of each cell line was analyzed using MiXCR . The 936n = 25), mantle cell lymphoma (n = 4), Burkitt lymphoma (n = 10) and chronic lymphocytic leukemia-small lymphocytic lymphoma  etc. High expression of both IGH (heavy chain) and IGK/L (light chain) rearrangement are detected in all of the mantle cell lymphoma, Burkitt lymphoma and CLL-small lymphocytic lymphoma cell lines , except two , express high levels of clonal rearrangement of both IGH and IGL/K  and CCLE. The analysis of the expression of major lineage genes also suggests that these two cell lines should not be grouped together with other DLBCL cell lines, as none of them express any typical B cell genes such as CD19, CD20 or CD79. Notably, cell line A4/Fuk also displays an abnormal expression pattern of major lineage specific genes. Conclusions of experiments reached from this cell line for DLBCL research may also need to be interpreted with caution.Potential subclonality was also found in diffuse large B cell lymphoma (DLBCL) cell lines Pfeiffer and Toledo  also express high level of heavy and light chain BCR rearrangements. Cell line BCP-1 shows an inconsistent gene expression pattern as compared with other B cell lymphoma lines , suggesting that it might need to be reclassified into other blood cancer types. In contrast to the finding of subclonality in Burkitt lymphoma or DLBCL, cell lines NU-DUL-1 and JM1 contain more than 3 different IGLV rearrangements  were derived from either mature B cells  or post GC B cells), but are also known to have a global down-regulation of B cell gene expression and a general loss of B cell phenotype [Hodgkin lymphoma cell lines (henotype . Consistn = 15) express low levels of IGH rearrangement as their dominant clonotypes. Consistent with the notion that most of the ALL cells had arrested in early stages of B/T cell development [In contrast to the aforementioned cell lines arising from mature B cells, most of the B-ALL cell lines  with an identical CDR3 sequence . This disease is a neoplasia of T lymphocytes often possessing helper/inducer cell surface phenotype . ExpressWith the exception of the cell line ALL-SIL, which has a TCR gamma rearrangement (TRGV7-TRGJ1), the other 14 T-ALL cell lines express TCR beta rearrangements as their dominant clonotypes (TRBV-TRBD-TRBJ or TRBV-TRBJ). Most of the T-ALL cell lines express biallelic-rearrangements –30 or pon = 31; blast phase CML, n = 12; CML, n = 2; essential thrombocythaemia, n = 1), less than 50–100 BCR/TCR reads were detected in the dominant rearrangement (CPM < 0.5). A few rearrangements appear to be commonly detected in a number of solid cancer and AML cell lines with very low read counts. For example, the rearrangements TRAV8–7 -TRAJ19  were commonly found in many cell lines with extremely low read count (< 30). These barely expressed rearrangements may be caused by non-specific sequencing noise or possibly due to the trace amount of cross contamination during sample preparation and sequencing [Cell lines derived from solid tumors and myeloid cells do not undergo B/TCR rearrangements. In almost all of the solid tumor cell lines (except for the few which would be discussed here) and myeloid derived cancer cell lines  carry clonal rearrangements . This cell line was derived from a mucinous gastric adenocarcinoma from a 52-year-old Japanese male . ConsideThe same bioinformatics pipeline was used to analyze 462 EBV transformed normal B lymphocyte cell lines which were immortalized from healthy donor’s B cells. Rearrangements of both IGH and IGK/L were readily detected in almost all of the lymphocyte cell lines Fig. . IGH reaWe hypothesize that EBV mediated transformation occurred in a number of B cells, and during culture, some clones outcompeted the others and gradually became dominant clones. Therefore, diversity was lost and the population became oligoclonal or even monoclonal , 40. In https://www.genecards.org). In addition, the feasibility of profiling BCR repertoires from RNA sequencing data have recently been explored in CLL [https://www.ncbi.nlm.nih.gov/sra). This suggests that the BCR/TCR repertoire pattern may be used as an alternative/complement authorization method for lymphocyte derived cell lines. The advantage of this approach is that when RNA sequencing data is available , the cell lines (need to be lymphocyte derived cells) used in these studies can be simultaneously validated based on the BCR/TCR profile. As such, the RNA sequencing data deposited in the online public database  can be further checked and authenticated independently by any researchers. Crucially, we noticed that poly A selection before sequencing is important for examining the BCR/TCR repertoire, as poly A enriched samples generated 100-fold more BCR/TCR sequencing reads as compared to samples which were not subjected to  poly A selection but were sequenced at similar sequencing depth. Nonetheless, RNA sequencing of samples without poly A selection can still discover the majority of BCR/TCR rearrangements. However, accuracy of clonotype fraction of subclonal rearrangements may be significantly affected.One of the potential pitfalls of profiling the BCR/TCR repertoire using RNA sequencing is that non-transcribed rearrangements may not be detected, and the clonality analysis may be biased by the expression level of BCR/TCR loci. Nevertheless, in comparison to traditional PCR based method which is labor intensive and requires well trained staffs, NGS/RNA sequencing based analysis is more standardized and can be simply outsourced to big sequencing centers. In contrast to PCR based analysis which is limited by the number of loci that can be feasibly examined, NGS based profiling can comprehensively detect all expressed TCR/BCR rearrangements, including some rearrangements which are not usually included in PCR based analysis . The BCR/TCR repertoire identified using RNA sequencing were consistent with previous studies using BIOMED-2 PCR Sanger sequencing , 30 . However, in the B cell lymphoma cell lines NU-DUL-1, JM1 and the Multiple myeloma cell line AMO-1, more than three different IGL  or IGH (cell lines AMO-1) rearrangements were detected, suggesting independent biclonality in these cell lines. As cancer cell lines are generally regarded as of monoclonal origin, the potential biclonality at these cell lines is interesting and may require further detailed study. On the other hand, our observation of high clonality and potential clonal selection/evolution in EBV transformed normal B lymphocytes suggests that careful experimental design and interpretation of the result may be required when using EBV transformed lines as a model to study normal B cell population, B cell gene expression or quantitative trait loci (QTL) , 46.In summary, we comprehensively profiled the B/T cell receptor repertoire in 936 cancer cell lines and 462 samples of EBV transformed normal B lymphocytes. The relative “pure” feature of cancer cell lines circumvents the problem of tumor infiltrating T/B and stroma cells in primary tumor samples. Our analysis provides unique insights into the BCR/TCR rearrangement repertoire and clonality of cell lines derived from lymphocyte cells.Additional file 1:Figure S1. Expression of CD19/CD20, CD79A/B and RAG1/RAG2 in blood cancer cell lines. X axis: 164 blood cancer cell lines grouped base on the disease types (rectangle color bar below the X axis), each dot in X axis represent one cell line, and the Y axis, expression level  of indicated genes. Figure S2. Clonal fraction (filtered by >30 reads) of B-ALL, multiple myeloma, diffuse large B cell lymphoma, Burkitt lymphoma, B cell lymphoma (unspecified) and mantle cell lymphoma base on IGH or IGHK/L. Blue color indicates the clonotype fraction of the most dominant clone, Red color indicate the clonotype fraction of second dominant clone, Yellow color indicate the third dominant clone, while any smaller subclones were aggregated and labelled in grey. Figure S3. Clonal fraction (filtered by >30 reads) of T-ALL and anaplastic large cell lymphoma base on TRCA or TRCB. Blue color indicates the clonotype fraction of dominant clone, red color inidicate the clonotype fraction of second dominant clone, while gray color indicate the third dominant clone. Figure S4. Heatmap showing the usage of IGK/L V genes (A), IGK/L J genes (B), and constant region (C) in 462 samples of EBV transformed normal B lymphocytes. Figure S5. The phylogenetic tree inferred based on the rearrangement of the CDR3 region of IGH, IGK and IGL of EBV transformed B lymphocyte samples ERR188025, ERR188358 and ERR188212. These three cell lines have much higher number of rearrangement types than the other B lymphocyte lines. Clonal Fraction (upper panel) and the read counts (lower panel) of the dominant clone of 462 samples of EBV transformed normal B lymphocytes. (ZIP 1290 kb)


Ferula L. includes perennial flowering plants belonging to the Apiaceae family. This genus is a rich source of biologically active phytochemicals such as sulfur-containing derivatives, coumarins, sesquiterpenes, sesquiterpene lactones, sesquiterpene coumarins, glucuronic acid, galactose, arabinose, rhamnose, and daucane esters. Over the last decade, considerable attention has been paid to biological activities of these compounds; it is assumed that the most prominent biological features of the genus Ferula are their cytotoxic effects. This article discusses cytotoxic activity of the genus Ferula and their important compounds.The genus Ferula and their important compounds, the type of cell line used in vitro, concentrations of the extracts/active compound that were used, and the underlying mechanisms of action through which Ferula-related chemicals induced cytotoxicity. In addition, we explained different mechanisms of action through which the active constituents isolated from Ferula, could decrease cellular growth.In this mini-review article, papers published from 1990 to April 2016 were included and the following information was discussed; cytotoxic activity of the genus Ferula plants and found to be appropriate as adjuvant therapy for certain diseases, should be identified. Also, the versatile biological activities of sesquiterpene coumarins suggest them as promising agents with a broad range of biological applications to be used in the future.It is highly recommended that potent and effective compounds that were isolated from  Ferula includes perennial flowering plants belonging to the family Apiaceae (Umbelliferae). This genus consists of about170 species which are distributed worldwide. Out of 30 species of Ferula that could be found in Iran, 16 plants are endemic. Different species of the genus Ferula are broadly distributed in arid areas from the eastern Mediterranean regions to central Asia  Ferula species, showed cytotoxic activity by inhibition of the growth of human M4Beu metastatic pigmented malignant melanoma cells through induction of cell cycle arrest in G1 and caspase-dependent apoptosis   showed cytotoxic activities against human melanoma cell line SK-MEL-28  cells . Similar results were found for feselol, a sesquiterpene coumarin isolated from the fruits of F. badrakema , IGROV-1 and OVCAR-3 (human ovarian cancer cell line), respectively  against MCF-7 and K562 cancer cells F. oopoda showed significant cytotoxicity. For example, dehydrooopodin revealed significant cytotoxicity with IC50 values of 5 and 15 µM against K562 and MCF7cancer cell lines, respectively , K562 (leukemia), MCF7, and COLO 205 (coloncarcinoma) cell lines, as well as the multidrug-resistant human cancer cell lines KB-C2 (colchicine-resistant KB) and K562/ADR (Adriamycin-resistant K562). These compounds showed moderate cytotoxicity with IC50 values ranging from 24.7 to 56.9 μg/mL F. gummosa extract has specific cytotoxic effects mainly against MCF7 and oral cancer cell lines  and one normal (MDBK) cell lines. In another study, the cytotoxicity of some of the Iranian medicinal Ferula species was examined and all the extracts and oleo-gum resins of F. assa-foetida showed dose-dependent cytotoxicity , cancer cell lines. They revealed that hexane and chloroform fractions of these plants have cytotoxic effects at concentration up to 100 μg/ml. They also reported that the cytotoxicity of F. persica extracts was higher than that of F. hezarlalezarica extracts  F. assa-foetida extract displayed neuroprotective effects in a glutamate-induced neurotoxicity model  was reported  showed that this activity is dependent on the type of solvent (methanolic>ethanolic) and its concentration (higher methanolic content>lower methanolic content)  and 800 mg/mL  were significantly decreased. Also, the F. gummosa root extract reduced the viability of GP-293 cells at concentration of 750 mg/mL  Some other cell-based assaysF. szowitsiana revealed cytotoxic properties. Umbelliprenin showed the highest inhibitory activity against M4Beu melanoma cell line (IC50=12.4±0.5 µM) compared to cisplatin (23.1±0.8 µM)   exhibited tubulin-polymerizing activity. Under Ca2+-free conditions, ferulenol appeared to be equipotent as Taxol in promoting tubulin assembly  Ferula, showed apoptosis-inducing activities in cancerous cells by induction of sub-G1 peak as revealed by PI staining . Farnesiferol C could be considered a potential chemotherapeutic agent; its anticancer effects are partly mediated via inducing tumor cells apoptosis by increasing the Bax/Bcl-2 ratio . They revealed that auraptene induced a sub-G1 peak in the flow cytometry histogram of treated cells compared to control cells. In this study, DNA fragmentation was suggested as one of the underlying mechanisms of auraptene-induced apoptosis. Also, western blot analysis showed that auraptene significantly up-regulated Bax expression in MCF-7 cells compared to untreated controls F. ferulaeoides (Steud.) Korov. Induced C6 glioma cell apoptosis and endoplasmic reticulum (ER) stress, via mitochondrial and death-receptor-mediated pathways  . They also reported that F. gummosa extracts inhibited AGS cell line proliferation in a dose-dependent manner with IC50 values of 37.47 µg/mL for flower and 32.99 µg/mL for leaf extracts. F. gummosa extracts also induced apoptosis, as reflected by DNA fragmentation and plasma membrane translocation of phosphatidyl serine , reflecting the possible presence of anti-angiogenic compounds .The ethyl acetate fraction of Mechanisms of actionFerula genus, showed both growth inhibitory and cytotoxic activities in different cancerous cell lines  through cell cycle arrest in G1 phase -induced proliferation and inhibited VEGF-induced migration and tube formation in human umbilical vein endothelial cells (HUVECs). These effects were accompanied by decreased phosphorylation of p38-mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and AKT, and decreased expression of VEGFR targets endothelial nitric oxide synthase (eNOS) and cyclin D1 in VEGF-treated HUVECs  plants and their main constituents. This review aimed to highlight cytotoxic activities of Ferula species and their phytochemicals . We also It is assumed that the most prominent biological features of the genus Ferula are their cytotoxic effects. Previous reports proposed that Ferula phytochemicals have different activities. This probably suggests that much effort still remains to be made to identify potent and effective Ferula compounds that could be appropriate to be used as adjuvant therapy along with the conventional antibiotics. It is ultimately suggested that considering the versatile biological activities of sesquiterpene coumarins, these compounds may have an even broader range of biological applications in the future.


Treatment results for multiple myeloma and plasma cell leukemia have considerably improved, but cure remains elusive and establishing new therapeutic approaches constitutes a major unmet clinical need. We analyzed the anti-myeloma properties of the aza-anthracenedione pixantrone which has been successfully used in a phase III study for the treatment of patients with aggressive non-Hodgkin’s lymphoma as monotherapy as well as in combination regimes in vitro and in an adapted in vivo model  assay). Pixantrone significantly inhibited proliferation and metabolic activity of all investigated myeloma cell lines. Importantly, anti-myeloma effects were more pronounced in tumor cell lines than in stromal cells, mesenchymal stem cells, and peripheral blood mononuclear cells of healthy controls. Apoptosis of myeloma cell lines was observed only after a 7-day incubation period, indicating a fast cytostatic and a slower cytotoxic effect of this drug. Pixantrone reduced the viability of primary plasma cells of patients and induced downregulation of myeloma-cell growth in the CAM assay. Additionally, we demonstrate in vitro synergism between pixantrone and the histone deacetylase inhibitor panobinostat with respect to its anti-proliferative features. From these data, we conclude that systematic investigations of the clinical usefulness of pixantrone in the framework of controlled clinical trials are clearly indicated .The online version of this article (10.1007/s00277-019-03797-6) contains supplementary material, which is available to authorized users. Multiple myeloma (MM) is an incurable malignant disease of immunoglobulin and/or free light chains producing monoclonal plasma cells, clinically characterized by hypercalcemia, renal failure, anemia, and osteolytic bone disease . Considerable clinical progress has been made with respect to higher response rates, better remission-induction efficiency and depth, as well as the improvement of both progression-free and overall survival. However, cure remains an elusive therapeutic goal. Nearly all patients will sooner or later relapse and develop treatment resistance to all available drugs and treatment modalities . Median Plasma cell leukemia (PCL) is a leukemic variant of myeloma arising either de novo or from clinically pre-existent MM in mostly heavily pretreated patients and carries an even worse prognosis than does relapsed/refractory MM. Treatment options are limited, and no drug has ever been explicitly registered for this indication. Furthermore, there is no generally accepted standard of care, although most hematologists use—depending on previous treatments—combinations of MM drugs, steroids, and chemotherapy . AnthracPixantrone (PIX) is structurally similar to the anthracycline doxorubicin (Dox), but with an improved toxicity profile, especially with regard to cardiotoxicity , 12, whiHere, we investigated the anti-myeloma properties of PIX by (1) analyzing its effects on myeloma cell lines, (2) screening in vitro for anti-myeloma synergisms with putative combination partners, (3) confirming its anti-myeloma effect in a chorioallantoic membrane assay (CAM), and finally (4) generating confirmatory evidence with primary patient material.All patients gave written informed consent to the use of their biologic materials for research (EU FP7 consortium OPTATIO) and documentation of their clinical data via the Austrian Myeloma Registry (AMR) after extensive discussion of the respective procedures.Patient disposition is outlined in Table 2. The cells were serially passaged twice a week. All cell lines were authenticated by typing short tandem repeats. Mycoplasma contamination was routinely monitored and only mycoplasma-free cultures were used. Mesenchymal stem cells were purchased from PromoCell  and cultured in Mesenchymal Stem Cell Growth Medium, according to the manufacturer’s instructions.Myeloma cell lines ) and the stromal cell line HS-5  were cultured in RPMI 1640 without phenol red, supplemented with L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and fetal calf serum . Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO4) were incubated with 1 μg/mL phytohemagglutinin  and appropriate concentrations of PIX for 72 h.PBMC from two patients with de-novo PCL, three patients with secondary PCL, and two patients with relapsed/refractory MM as well as four healthy volunteers were isolated by Ficoll density gradient centrifugation. To generate activated PBMC cells -2,5-diphenyltetrazolium bromide (MTT) assay  according to the manufacturer’s instructions. To exclude unspecific staining by FCS-containing medium, the optical density (OD) of the respective medium was subtracted. Metabolic activity in the absence of the compounds was set at 100%.Induction of programmed cell death for cell lines and primary cells after different time periods was determined by staining with Annexin V (Ann V)-FITC-labeled antibody and propidium iodide (PI). Samples were further analyzed by flow cytometry  and data were evaluated utilizing FlowJo software .The CAM assay used in this study was essentially performed as described previously . In detap value < 0.05 was considered statistically significant. Additive and synergistic effects of drugs were defined according to the following formula: additive mode of action: surviving fraction (SF) (compound A + compound B) = SF (A) × SF (B); synergistic mode of action: SF (A + B) < SF (A) × SF (B) [The Wilcoxon rank-sum test was used to analyze the differences between proliferation, metabolic activity, and apoptosis induction in the absence and the presence of the respective concentration of the compounds . A × SF (B) .Proliferation of myeloma cell lines was determined after 72 h of incubation with various concentrations of PIX. All cell lines tested showed reduced proliferation, and the extent was dose-dependent, starting at 0.05 μM PIX Fig. . At a PIAs PIX structurally resembles Dox, a drug still widely used for MM treatment, the anti-proliferative capacity of Dox was analyzed in parallel in selected myeloma cell lines after a 72-h incubation. As shown in Fig. Similar anti-proliferative effects of PIX were observed in a co-culture system with the stromal cell line HS-5 . Apoptosis induction, however, was observed only after a 7-day incubation  in combination with various “new” anti-myeloma compounds such as bortezomib (5 and 10 nM), panobinostat (3 nM), and lenalidomide (10 and 25 μM) and “classical” MM drugs such as Dox (10 and 50 nM), bendamustine (10 and 50 μM), and dexamethasone . The most effective combination was PIX (50 nM) and panobinostat  Fig. . This inAs already shown for primary cells, PIX (50 nM) and Dox (50 nM) revealed also synergistic effects in the myeloma cell lines suggesting additional mechanisms of these drugs. Similar strong anti-proliferative activity was observed with PIX (50 nM) and bendamustine (10 μM).PIX is approved in the EU for the treatment of aggressive non-Hodgkin’s lymphoma patients. We therefore investigated if PIX might also be effective in MM and PCL.In all cell lines tested, relatively low PIX concentrations (between 0.1 and 0.25 μM) already significantly inhibited cell proliferation. Importantly, a concentration of 0.25 μM corresponds to plasma levels achievable with standard-dose PIX in patients . This loImportantly, anti-myeloma effects were more pronounced in myeloma cell lines than in the stromal cell line HS-5, MSC, and activated PBMC of healthy controls. The results of the CAM assay further support the putative anti-tumor specificity of PIX while inducing no toxic effect on the embryo.Inhibition of metabolic activity showed a more variant pattern and may reflect the clinical and biological heterogeneity of this disease. The inhibition of metabolic activity after a 3-day culture period was accompanied by apoptosis induction of the MM cell lines after 7 days only, which, nevertheless, is in accordance with the literature .In direct comparison to Dox, higher PIX concentrations were necessary to induce similar strong effects in myeloma cell lines as well as in primary patient cells. However, the improved toxicity profile of PIX , 11 arguWe were able to demonstrate an impressive in vitro synergism between PIX and PAN with respect to their anti-proliferative features on myeloma cell lines. PAN is a potent, oral pan-deactylase inhibitor that increases acetylation of proteins involved in multiple oncogenic pathways . It has Similar strong combinatorial activity was observed with Dox (in cell lines as well as in primary cells) and bendamustine. As at least bendamustine is a good therapeutic option in myeloma therapy—especially in renal failure—another interesting option to be investigated might thus be a bendamustine-PIX combination , 28.In conclusion, PIX induced a strong and fast cytostatic effect in myeloma cell lines and induced apoptosis after a longer incubation period suggesting a putative role of PIX as part of possible combination therapies in relapsed/refractory MM and PCL patients in the framework of controlled clinical trials.Supplementary Figure 1(DOCX 18 kb)


Drug delivery systems are molecular platforms in which an active compound is packed into or loaded on a biocompatible nanoparticle. Such a solution improves the activity of the applied drug or decreases its side effects. Dendrimers are promising molecular platforms for drug delivery due to their unique properties. These macromolecules are known for their defined size, shape, and molecular weight, as well as their monodispersity, the presence of the void space, tailorable structure, internalization by cells, selectivity toward cells and intracellular components, protection of guest molecules, and controllable release of the cargo. Dendrimers were tested as carriers of various molecules and, simultaneously, their toxicity was examined using different cell lines. It was discovered that, in general, dendrimer cytotoxicity depended on the generation, the number of surface groups, and the nature of terminal moieties . Higher cytotoxicity occurred for higher-generation dendrimers and for dendrimers with positive charges on the surface. In order to decrease the cytotoxicity of dendrimers, scientists started to introduce different chemical modifications on the periphery of the nanomolecule. Dendrimers grafted with polyethylene glycol (PEG), acetyl groups, carbohydrates, and other moieties did not affect cell viability, or did so only slightly, while still maintaining other advantageous properties. Dendrimers clearly have great potential for wide utilization as drug and gene carriers. Moreover, some dendrimers have biological properties per se, being anti-fungal, anti-bacterial, or toxic to cancer cells without affecting normal cells. Therefore, intrinsic cytotoxicity is a comprehensive problem and should be considered individually depending on the potential destination of the nanoparticle.  Dendrimers, also known as starburst polymers , cascadeFirstly, dendrimers are characterized by a compact structure and a strictly predictable molecular weight (MW). They are monodisperse, polyvalent, usually globular macromolecules with a regular and highly branched three-dimensional architecture. They consist of a central core molecule surrounded by branches called dendrons, and are terminated with functional surface groups. The essential feature of these nanomolecules is their generation, defined as the number of layers attached to the core and denoted as Gn, where n can range from 0 to 12. All these properties make dendrimers excellent candidates for nanomedical applications. As compounds that are so intensively studied for their potential use in biomedicine, dendrimers should meet several criteria. Specifically, they should be non-toxic, non-immunogenic, biopermeable (possess the ability to cross biobarriers), able to stay in blood circulation until the desired effects occur, and able to target specific biological structures. Many groups of dendrimers fulfill most of these requirements; however, their utilization in biomedicine is often limited due to their high cytotoxicity . This rePoly(amido amine) (PAMAM) and poly(propylene imine) (PPI) dendrimers are the two most commonly studied dendrimers, probably due to their broad commercial availability . However2 dendrimer through electrostatic interactions between the dendrimer amines and the carboxyl group of the drug. The complexed drug entered A549 cells much more rapidly than the pure drug, suggesting that dendrimers were able to carry the complexed drug inside cells efficiently [Boas et al. showed that thiourea-functionalized PPI dendrimers can be used as a drug delivery system. The encapsulation properties of these dendrimers are based on the formation of dense, hydrogen-bonded surface shells with solid-state characteristics. Small guest molecules captured in the interiors of the dendrimers were unable to escape even after extensive dialysis. The number of encapsulated guest molecules was directly proportional to their size and shape. Such a dendrimer box could be opened controllably by partial or total hydrolysis to release either some or all of the entrapped guest molecules . In addiiciently . Anothericiently . Interesiciently ,20. Khaniciently , while Giciently . Photodyiciently .Dendrimers can be used as therapeutic factors in neurodegenerative disorders. In vitro studies showed that dendrimers were capable of interfering with the formation of amyloid fibrillar structures typical to the onset and development of neurodegenerative disorders, such as Alzheimer’s, Parkinson’s, and prion diseases. Wasiak et al. demonstrated that cationic phosphorus dendrimers of generations 3 and 4  were able to affect β-amyloid and MAP-Tau protein aggregation processes. Assessment of the cytotoxicity of formed fibrils and intermediate products during Aβ 1–28 aggregation was performed using a neuroblastoma cell line (N2a). The results indicated that CPDs were able to reduce the toxicity of aggregated forms of Aβ 1–28 . SimilarCandida albicans. It was shown that maltose-modified PPI dendrimers displayed the highest antibacterial activity and a striking selectivity toward a Gram-positive bacterium, Staphylococcus aureus. Simultaneously, these nanomolecules exhibited insignificant toxicity toward eukaryotic cells [®, was developed by Starpharma . Preclinical development studies demonstrated that VivaGel® was 100% effective at preventing infections of primates exposed to a humanized strain of simian immunodeficiency virus (SHIV). In 2012, the company started crucial phase III clinical trials for the treatment of bacterial vaginosis. First, VivaGel®-containing condoms were approved on the Australian market. Then, the product was approved for the treatment and prevention of bacterial vaginosis (BV) and the prevention of sexually transmitted infections [Dendrimers and dendrimer complexes proved to be effective antipathogenic agents in vitro. Unmodified and maltose-modified PPI dendrimers were tested for their potential against a set of pathogenic bacteria and yeast—ic cells . Moreoveic cells . These cic cells ,34. PAMAic cells . One of ic cells . The PLLfections .Plasmodium falciparum T- and B-cell stimulatory peptides, is a good example of such a system [Many researchers aimed to use dendrimers for vaccine and immunization purposes. A multiple antigenic peptide (MAP) system, based on PLL dendrimers, was pioneered by Tam et al. in the late 1980s ,39. It ca system . Furthera system .Since the pioneering work of Lauterbur, Wiener, and Tomalia et al. , numerouMany studies focused on the utilization of amino-terminated PAMAM or PPI dendrimers as non-viral gene transfer agents, increasing the transfection efficiency ,45. DendInterestingly, dendrimers were also investigated for their use in biomedical applications as artificial proteins. Owing to their nanoscale dimensions, globular shape, and other properties, they can mimic many important proteins. For example, the size of insulin (3 nm) is comparable to the size of a PAMAM G3 dendrimer. Similarly, cytochrome C (diameter = 4 nm) and hemoglobin (diameter = 5.5 nm) correspond to the sizes of PAMAM G4 and G5, respectively. A comprehensive strategy for designing dendritic nanostructures to mimic globular proteins was described by Tomalia et. al. . They syDendrimers possess great biomedical potential and, as presented in The cytotoxicity of dendrimers depends strongly on the number and nature of functional surface groups. Cationic dendrimers often exhibit high toxicity, whereas anionic and neutral dendrimers show slight or no toxic effects. For example, poly(amido amine) (PAMAM) and poly(propylene imine) (PPI) dendrimers possessing terminal primary amines are characterized by concentration- and generation-dependent toxicity ,50, wherVibrio fischeri, Daphnia magna, Thamnocephalus platyurus, and two fish cell lines. The toxicological response correlated well with the dendrimer generation and, therefore, with the particle surface area; an increase in surface area led to an increased toxic response. Daphnia magna was found to be the most sensitive model, and the RTG-2 fish cell line was the least sensitive [Mukherjee et al. published an interesting paper, disclosing an indirect impact of PAMAM dendrimers on cell viability. The authors observed generation-dependent toxicity of PAMAM G4, G5, and G6 to human keratinocytes (HaCaT) and primary colon adenocarcinoma cells (SW480). Based on spectroscopic methods, and the measurements of the zeta size and potential of dendrimers in fetal bovine serum (FBS)-supplemented medium, it was found that dendrimers absorbed proteins from the culture media. These data suggested the indirect mechanism of PAMAM cytotoxicity resulting from the depletion of medium components . In the ensitive . Interesensitive . 2, COONa, COOH, PEO) on their in vitro behavior. Dendrimers with amino terminal groups displayed concentration- and generation-dependent hemolysis, while anionic and CSi–PEO dendrimers were neither hemolytic nor cytotoxic toward a panel of cell lines in vitro. Polyether dendrimers with carboxylate and malonate surfaces were not hemolytic at 1 h; however, after 24 h, unlike anionic PAMAM dendrimers, they were lytic [Several types of dendrimers  or diaminoethane (DAE) core, poly(ethylene oxide) (PEO)–grafted carbosilane (CSi–PEO), and polyether dendrimers) were compared regarding the influence of generation , and surface functionality  groups. Dose–response effects of both modified and unmodified PAMAM were examined on two cell lines—a fish cell line (RTG-2) and a rat hepatoma cell line (H4IIE). No toxic effects of amino-terminated PAMAM dendrimers were observed for both H4IIE and RTG-2 cells lines when the concentration was below 500 μg/mL. For surface-modified PAMAM dendrimers, higher cytotoxicity was found for the H4IIE cell line [As mentioned above, surface modification is a common way to increase the biocompatibility of dendrimers. For example, modified PAMAM dendrimers of generation G4 with 4-carbomethoxypyrrolidone surface groups (PAMAM–pyrrolidone dendrimer) showed only minor toxicity and no ability to induce apoptosis. The most important finding was the lack of influence of the PAMAM–pyrrolidone dendrimer on intracellular ROS level and mitochondrial membrane potential even at the highest tested concentration (200 µM) ,60. PAMAell line . Wen et ell line . In anotell line . The worell line . In theiell line . Janaszeell line . In anotell line . It seemThe ability of dendrimers to cross cell membranes is of much interest for their application in drug and gene delivery. Indeed, a safe alternative to the viral system used in gene therapy is a nonviral carrier, and dendrimers seem to be excellent for this role. Albertazzi et al. revealed that PAMAM dendrimers G2, G4, G6, and lipidated G4 possess similar properties to widely used cell-penetrating peptides (the arginine-rich motif derived from the HIV protein TAT). Furthermore, they are not toxic toward the HeLa cell line (1.5 μM concentration for 1 h) and, thanks to their chemical tunability, they represent an attractive option for drug and gene delivery . Also, CNavath et al. demonstrated the development of biocompatible PAMAM G4 dendrimers, enabling the attachment of drugs (indomethacin and dexamethasone) and imaging agents (dexamethasone and FITC) without using any specific linkers. This could be achieved thanks to multiple amino-acid-based orthogonal surface groups at the dendrimer periphery. Furthermore, one of the two functional handles at the periphery was used to develop in situ forming hydrogels, whereas the other handle could be used for conjugating drugs. An in vitro cytotoxicity test and a hemolysis assay showed that the heterobifunctional dendrimers were non-cytotoxic in the 100 ng/mL to 1 mg/mL concentration range . Krishna et al. reported the synthesis of several PPI dendrons and dendrimers, which were constructed by involving an ether as the linker component and an imine as a branching component. The adopted synthetic sequence allowed an alcohol, an amine, or a carboxylic acid to be installed at the peripheries. The carboxylic-acid-terminated dendrons and dendrimers were evaluated for their cytotoxic properties, and, while most dendrons and dendrimers did not exhibit any measurable cytotoxicity, even up to 100 μg/mL, the second-generation dendrimer with the benzenoid core exhibited mild toxicity at concentrations above 30 μg/mL . It was w/w) was achieved with etoposide, a hydrophobic anticancer drug. A cytotoxicity assay demonstrated that the star-PCL–PEG copolymer was non-toxic for cells, indicating that this type of block copolymer could be used as a drug delivery carrier [Wang et al. proposed the synthesis of a star polymer composed of amphiphilic block copolymer arms. The core of the star polymer was a PAMAM dendrimer, the inner block of the arm was lipophilic poly(ε-caprolactone) (PCL), and the outer block of the arm was hydrophilic PEG. The star-PCL polymer was synthesized first via ring-opening polymerization of ε-caprolactone with a PAMAM-OH dendrimer as an initiator. The PEG polymer was then attached to the PCL terminus via an ester-forming reaction. It is known that hydrophobic dyes and drugs can be encapsulated in the micelles. A similar effect was observed for the star polymer. A loading capacity of up to 22% ( carrier .Cyprinus carpio (EPC) [50 values ranging from 0.3 to 1.6 μM. Interestingly, the complexation of the terminal ligands of 1G3 dendrimers by copper(II) metal strongly increased IC50 values in non-cancer cell lines [Lazniewska et al. studied toxic responses of two low generations of cationic phosphorus dendrimers (CPDs) of generation 2 and generation 3 toward murine embryonic hippocampal cells (mHippoE-18) and N2a cells. The obtained results showed that CPDs G3 were highly cytotoxic at concentrations above 1 μM and at 0.7 μM for mHippoE-18 cells. A significant decrease in cell viability corresponded to severe changes in cellular processes, such as massive ROS generation. Unlike other cytotoxic dendrimers, which activated the apoptotic pathway in cells, CPDs caused a breakdown of cellular processes followed by a necrotic cell death . The cytio (EPC) . It was ll lines .Escherichia coli, and the toxicity against human cells was evaluated with the human epithelial cell line A549. The silver particles, especially those prepared from dendrimers of higher generations, demonstrated a significant antibacterial effect against E. coli. No toxicity against human cells was observed for the silver particles even in case of the highest investigated silver concentration. Therefore, such a construct could offer great potential for application as an antibacterial agent with low human toxicity [Zeng et al. presented hyperbranched polyester Boltorn decorated with PEG groups, which showed potential as an efficient and specific delivery platform for drugs and imaging agents. The cytotoxicity, cellular uptake profiles, and intracellular trafficking of polymer micelles were tested in MDA-MB468 breast cancer cells. The uptake of these nanoparticles was positively correlated with time and concentration and was energy dependent. These nanoparticles were shown to be internalized into cells via clathrin- and macropinocytosis-mediated endocytosis. Moreover, they did not significantly affect the viability of the cells . Hydroxytoxicity . Feliu etoxicity . The biotoxicity . One more architecture characterized by improved biological properties is dendritic polyglycerol (dPG). Various dPG derivatives possessing neutral, cationic, and anionic charges were compared with amine- and hydroxyl-terminated PAMAM dendrimers. In the U-937 cell line, dPG with terminal hydroxyl groups, dPG sodium sulfate, dextran, and linear PEG caused no toxic effect compared to dexamethasone used as a reference standard. Similarly, amine terminal dPGs (7% and 18% amines) were also almost non-toxic . On the other hand, three polymers, namely, dPG with higher amine functionality (45% and 100%), PAMAM G4 hydroxyl and amine dendrimer, and PEI, caused higher cell toxicity than the uncharged hydroxyl dPGs, dextran, and PEG. The cell compatibility results showed that the dendritic polyglycerols were as safe as linear PEG polymer or dextran, which indicates the suitability of dPG derivatives in delivering therapeutic agents .tert-butoxycarbonyl (Boc) or benzyloxycarbonyl (Cbz) protecting groups, Boc-protected or unprotected natural amino acid residues, ethylene-diamine ligands, and/or dansyl fluorescence labels. The cytotoxicity was determined in vitro in concentration-dependent assays using the MCF-7 cell line. The internal structure of the presented dendrimers did not seem to play a profound role in cytotoxicity, despite the common view that the interior of low-generation dendrimers is accessible. The surface decoration, however, was crucial for toxic effects. Most of the examined non-charged dendrimers  were non-toxic, although they were clearly bioavailable as proven by cell uptake experiments, and all completely diaminopropionic-acid-decorated dendrimers (positively charged) were also non-toxic. These results, especially the latter case, indicated that positive charges on a dendrimer surface did not always lead to cell toxicity and that another structure/toxicity correlation might also play a role [Fuchs et al. developed a new series of dendrimers G1 and G2 possessing various surface functionalities. Terminal amine groups of these dendrimers were decorated with protons, y a role . Huang et al. also described synthesis of two non-peptidic fluorescently labeled Newkome-type dendrimers, differentiated over a varied alkyl spacer with guanidine end moieties. These novel nanomolecules were designed as biocompatible carriers of bioactive cargo able to cross the cell membrane and localize in targeted cell compartments. The behavior of dendrimers was comparable in two tested cell lines: NIH-3T3 fibroblasts and human microvascular endothelial cells (HMEC). The differential localization patterns of the two molecular transporters could be controlled through the variation of alkyl spacer length at the terminal generation of the dendrimer. Intracellular delivery of bioactive entities into specific subcellular locations utilizing this practical approach might overcome limitations in drug delivery .Although cytotoxicity is usually the limiting factor for dendrimer application, it plays a more comprehensive role in clinical practice. Dendritic polymers chosen for the potential treatment of infectious diseases should be able to kill a pathogen while remaining harmless for a patient. Similarly, in anti-cancer therapy, dendrimers should affect abnormal cells only. This effect can be achieved with proper dose selection and by utilizing specific ligands or effects such as “enhanced permeability or retention”. This section reviews recent research on the cytotoxicity of dendrimers in the context of their potential clinical applications.Staphylococcus aureus and Staphylococcus epidermidis, Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, and yeast, Candida albicans. Cytotoxicity of all tested dendrimers was checked on B14, human liver hepatocellular carcinoma (HepG2), N2a, and rat liver cell lines (BRL-3A). The obtained results indicated that unmodified PPI dendrimers and PPI dendrimers modified by 25% maltose displayed antibacterial activity and a striking selectivity toward S. aureus, at the concentration of 1 µM, which, at the same time, was harmless for the eukaryotic cell lines [S. aureus) and Gram-negative (E. coli) bacteria, as well as against a fungal pathogen . However, contrary to the PPI dendrimers, lysine-based peptide ones exhibited some level of toxicity, although most of them caused only a slight (ca. 20%) decrease in B-14 cell viability for minimum inhibitory concentrations. The study also showed that the degree of branching, steric distribution, and types of hydrophobic (aromatic) groups and cationic centers are important components of dendrimeric structure and influence both antimicrobial potency and toxicity [PPI dendrimers were investigated for their cytotoxic and antibacterial activities by Felczak et al.; original PPI G4 dendrimers and PPI G4 with a surface modified by 25% and 100% maltose  were evaluated for their antibacterial activity against Gram-positive bacteria, ll lines . Klajnertoxicity . Bacillus subtilis and S. aureus (Gram-positive), as well as E. coli and Salmonella typhi (Gram-negative). Metallodendrimers also showed anti-tumor activity against the MFC-cell line. Pt(II)-containing metallodendrimers were found to be more efficient in the induction of MFC-7 death than Pd(II)-containing metallodendrimers, and both showed lower cytotoxicity than cisplatin (standard drug) [n = 1–3), were synthesized, and the pyrenyl group of the dendritic molecules was encapsulated in the arene ruthenium metallacages. The host–guest systems inhibited the growth of both cisplatin-sensitive and -resistant cancer cells (A2780 and A2780cisR). Moreover, these water-soluble host–guest systems showed cytotoxicity toward the cisplatin-resistant cell line that was comparable to free cisplatin in non-resistant human ovarian cancer cells [Metallodendrimers  are another group of dendritic compounds. It was revealed that polyamide metallodendrimers possess anti-bacterial activity against rd drug) . Pitto-Ber cells .Thiagarajan et al. characterized the activity of a PAMAM dendrimer conjugated with an anticancer drug camptothecin (PAMAM–CPT) on human colorectal carcinoma cells (HCT-116). The conjugate was stable under physiological pH (7.4) in phosphate-buffered saline (PBS) and in growth media (with 10% FBS) with minimal release of 4% and 6% drug, respectively, at 48 h. PAMAM–CPT inhibited the proliferation of HCT-116 cells and induced cell-cycle arrest with up to 68% of cells blocked in the gap 2 (G2) phase. Thus, the PAMAM–CPT conjugate was active against colorectal cancer cells in vitro, inhibiting their growth and inducing nuclear fragmentation. However, it would be useful to test the cytotoxicity of this conjugate against normal cells . p < 0.05) increased the cytotoxicity against both cell types. The conjugate had approximately 12-fold greater permeability across both apical and basolateral cell monolayers than that of PTX alone [Surface modifications of dendrimers were shown to be a factor enhancing the cell-penetrating ability of the drug–dendrimer complex, thus contributing to the inhibitory effect on cancer cell growth. Jia et al. modified PAMAM dendrimers G5 with acryloyloxyethyl phosphorylcholine (APC), a molecule possessing biomimetic properties. The hydrophobic interior of the modified dendrimer (G5-PC) was used to incorporate the anti-cancer drug adriamycin (ADR) and the G5-PC showed sustained release behavior for ADR. Cell viability and morphology analyses on the cancerous HepG2 cell line revealed that G5-PC was characterized by much lower toxicity in comparison to the unmodified PAMAM dendrimer. Furthermore, the drug-loaded G5-PC was shown to be internalized into cancer cells and could effectively decrease their viability . Teow etTX alone . PaclitaTX alone . In another study, PEGylated PAMAM dendrimer of generation 3 was loaded with fluorouracil (5-FU) and tested in vitro on the MCF-7 cell line. The results indicated that the PAMAM dendrimer presented higher toxicity as compared to its PEGylated counterpart. However, PEG-modified PAMAM dendrimer loaded with 5-FU exhibited antiproliferative activity against the cancer cell line. An additional benefit from the encapsulation of fluorouracil in the PEGylated dendrimer was a slow release profile of the drug. Moreover, in vivo studies revealed that the tested complex exhibited a significant decrease in the volume of the tumors, which were generated by the MCF-7 cancer cells .Despite their cytotoxicity, PPI dendrimers were also widely used as effective delivery vehicles for drugs. Wang et al. decreased the cytotoxicity of PPI dendrimers by acetylation and then used them to encapsulate anticancer drugs, including methotrexate sodium, sodium deoxycholate, and doxorubicin. The results indicated that the degree of acetylation determined the level of cytotoxicity and drug-loading capacity of the dendrimer. The drug-loading capacity of acetylated PPI dendrimers increased proportionally with the degree of acetylation on the dendrimer surface. PPI dendrimers with greater than 80% acetylation did not affect the viability of MCF-7 and A549 cells. The cytotoxicities of methotrexate sodium and doxorubicin were also significantly reduced when they were complexed with acetylated PPI dendrimers with high degrees of acetylation (>80%) owing to sustained drug release from the dendrimers. The results suggested that surface acetylation can reduce the cytotoxicity and improve the anticancer drug-loading capacity of cationic dendrimers . KesharwThe use of vector molecules for the targeted delivery of antitumor drugs provides selectivity for cancer cells. Yabbarov et al. used a recombinant receptor-binding fragment of alpha-fetoprotein (rAFP3D) as a vector molecule. The receptor of alpha-fetoprotein, a tumor marker, is expressed on the surface of many tumor cells, but not in normal human tissues. The vector rAFP3D was conjugated with a PAMAM G2 dendrimer and an anticancer drug, doxorubicin (Dox). The conjugate demonstrated a high cytotoxicity against human ovarian adenocarcinoma cell lines (Dox-sensitive SKOV3 cells and Dox-resistant SKVLB cells), while having low toxicity against human peripheral blood lymphocytes . AnotherZhang et al. synthesized a saccharide-terminated PAMAM G3 dendrimer, which was then conjugated with the drug methotrexate (MTX). This construct was shown to have potential as an anticancer nanodevice for the specific targeting and killing of folate receptor (FR)-expressing tumor cells. Their results showed that G3–MTX presented enhancement in binding avidity to folate-binding protein (FBP) that was three orders of magnitude higher than a free folic acid (FA), and internalized into FR-expressing KB cells  in a dose-dependent and receptor-mediated fashion . Han et Gupta et al. added DOX (approximately 26 and 65%) to the PPI dendrimers, as well as folate-conjugated PPI (PPI–FA) dendrimers. PPI–FA–DOX exhibited the highest percentage cell-growth inhibition compared to other formulations and to the drug itself. All three formulations showed a dose-dependent inhibition of MCF-7 cells. The higher uptake in the case of PPI–FA–DOX was possibly due to the ligand-specific targeting of dendrimers due to surface conjugation of the folic acid . The published toxicity levels of all dendrimers mentioned in this paper are summarized in Although there are active compounds that are able to inhibit or cure particular diseases, they are often not effective due to weak solubility, lack of ability to cross biological barriers, poor targetability, and sensitivity to the cell environment (pH) and its molecular machinery . A solution to this problem could be “packing” the active compound into or loading it onto a biocompatible delivery platform. One of the most promising options here is represented by dendrimers.Dendrimers, which were synthesized in the 1980s, quickly attracted attention of researchers from the biomedical field, and, in the 1990s, first works concerning their biocompatibility, cytotoxicity, and potential applications appeared. Dendrimers were tested as carriers of various molecules and, simultaneously, their toxicity was examined using different cell lines. It was discovered that, in general, toxic effects caused by dendrimers were dependent on the generation, the number of surface groups, and the nature of terminal moieties . A higher generation and an increased number of positive charges on the surface resulted in higher cytotoxicity. In order to decrease the cytotoxicity of dendrimers, scientists started to introduce different chemical modifications on the periphery of the nanomolecule. Dendrimers grafted with PEG, acetyl groups, carbohydrates, and other moieties did not affect cell viability, or did so only slightly, while still maintaining other advantageous properties. These properties include defined size, shape, and molecular weight, as well as monodispersity, the presence of the void space, tailorable structure, internalization by cells, selectivity toward cells and intracellular components, protection of guest molecules, and controllable release of the cargo. Intensive studies on dendrimers and their constant improvement resulted in their current status as systems of great potential in the biomedical field. In particular, they are developed as nanocarriers of drugs , nucleic acids in gene therapy , and imaging agents for MRI . Furthermore, some dendrimers also exhibit intrinsic biological activity, with anti-bacterial, anti-fungal, and anti-viral properties of dendritic nanomolecules previously demonstrated. Moreover, other dendrimers were reported to inhibit processes involved in neurodegenerative disorders, i.e., fibrillation and aggregation of specific proteins. Lastly, in several cases, it was demonstrated that dendrimers can be toxic to cancerous cells, while exhibiting no or low toxic effects toward normal cells. A synergistic effect of the combination of the active dendrimer and its cargo molecules (drugs) can result in a very potent therapy.The unique structure of dendrimers enables modifying the surface groups, which are largely responsible for properties of dendrimers, including cytotoxicity. However, it should be taken into account that diminishing cationic charge on the surface often reduces the therapeutic activity of dendrimers, e.g., the ability to create complexes with DNA or RNA. Therefore, partial modification is sometimes applied in order to achieve a balance between reduced toxicity and retained activity; this is frequently done via partial modification of PPI dendrimers by maltose or maltotriose .Depending on the specific medical application, a higher or lower dose of the dendrimer is needed. It allows finding the right dendrimer for a specific purpose from the large library of available dendrimers. Therefore, the toxicity of the dendrimers is not the main issue that slows down the process of commercializing dendrimers. However, this field certainly needs better consistency when testing the long-existing and new compounds. Overall, the intrinsic cytotoxicity of dendrimers can be overcome by precise chemical modifications. Their complexation with various bioactive molecules can be perfectly optimized, while being delivery-specific and safe for both the cell and the cargo. At present, dendrimers are an outstanding alternative to other drug carrier platforms by increasing the bioavailability and efficiency of transported compounds, thereby enhancing their therapeutic effects.