In general, SC are composed by a promising scaffold with great potential for the development of new drugs.Styrylchromones (SC) are a group of oxygen-containing heterocyclic compounds, which are characterized by the attachment of a styryl group to the chromone core. SC can be found in nature or can be chemically synthesized in the laboratory. As their presence in nature is scarce, the synthetic origin is the most common. Two types of SC are known: 2-styrylchromones and 3-styrylchromones. However, 2-styrylchromones are the most common, being more commonly found in nature and which chemical synthesis is more commonly described. A wide variety of SC has been described in the literature, with different substituents in different positions, the majority of which are distributed on the A- and/or B-rings. Over the years, several biological activities have been attributed to SC. This work presents a comprehensive review of the biological activities attributed to SC and their structure-activity relationship, based on a published literature search, since 1989. The following biological activities are thoroughly revised and discussed in this review: antioxidant, antiallergic, antiviral, antibacterial, antifungal, anti-inflammatory, and antitumoral, affinity and selectivity for A The chromone ring system or chromone core  . Over thH-chromen-4-one”. In the bibliographic search, 43 results were obtained, of which 4 were review articles (one of which was written in Japanese) and 39 were original articles. All the retrieved articles, written in English, from 1989 to the present (March 2021) have been analysed. From these articles, the cited references referring the biological activities of the SC were also included in this review. Furthermore, this review only includes studies with isolated compounds of synthetic and natural origin that are well characterized. Studies with SC's extracts or glycosylated derivatives were not included. Articles that do not present the methodologies used and with inconsistencies in the studied SC structures were also not included. The results presented throughout this review are presented according to the information available in the correspondent cited articles. Thus, the form of presentation of the results varies depending on the information available and may have the error associated (standard deviation or standard error of the mean) or not.The bibliographic search for this review was carried out in the PubMed database using the keywords “styrylchromone” and “styryl-4H-chromen-4-one  or 2-styryl-44-one 1, , where tne 1, 2,  are alsoHormothamnion enteromorphoides . Some of the 2-SC were more potent than the tested positive control, allopurinol (IC50 = 5.43 ± 0.08 μM). 2-SC 19 was the most active, with an IC50 value of 0.55 ± 0.03 μM, followed by 18, 14, and 17 . The structure of the most active compounds indicates that the presence of the catechol group (C-3′ and C-4′) on the B-ring of the derivatives (17-19) potentiates XO inhibition. The presence of two −OH groups linked to the A-ring also contributes to the inhibition, especially at C-5 and C-7 (19) leading to an increase in the activity when compared with the presence of just one −OH on the A-ring (17 and 18). This effect is also observed in the result obtained for 2-SC 14, which, despite having only −OH groups at C-5 and C-7 on the A-ring, proved to be a potent XO inhibitor , being more potent than the used positive control, rutin (IC50 = 3.66 ± 0.43 μM). The 2-SC from the other groups were less efficient  or completely ineffective . In summary, 2-SC from group 1 showed scavenging activity against all the detected ROS and RNS, while 2-SC from group 2 had a variable effect depending on the number and position of the −OH substituents on the A-ring, and for some reactive species, they did not show any activity. The activity of 2-SC from group 3 was generally low or inexistent. Therefore, for most of the reactive species studied, the catechol on the B-ring appears to play an important role in the scavenging activity. However, for HOCl scavenging activity, the −OH substituents on the A-ring assumed high importance, despite the existence of −OH substituents on the B-ring. The authors also tested some flavonoids structurally related to the tested 2-SC, which allowed to conclude that the styryl moiety also contributes to their observed outstanding antioxidant activity. A possible explanation for this fact might be the contribution of the styryl pattern to the stabilization of the radicals that are formed during the scavenging reactions  . Th. Th78–80 studies –39. 2-SCotype 1B  and compreported , 38.1, 5, 21, 56–58, 62, 70, and 81–83, in vitro, by a colorimetric assay, to determine the concentration of compounds that cause 50% cytotoxicity (CC50) and MNTC. Most of the tested 2-SC showed little toxicity, presenting a relatively large margin of security, at the highest concentration tested (150 μM). However, 2-SC 62, 70, and 83 showed a different toxicity profile in RAW cells, with 62 and 83 being the most toxic . Antiviral activity was evaluated by a plaque reduction assay in RAW cells. The results were presented as IC50 values and the selectivity index (SI) was also presented. 2-SC 5 and 58 were the most active  and presented high SI values . 2-SC 1, 57, 70, and 82 were less active but also demonstrated antiviral effects, and 21, 56, and 62 showed a weak anti-norovirus activity. In this study, the possibility of 2-SC 5 and 58 interfering in an early or late stage of MNV replication cycle was also evaluated. The antiviral effects of compounds decreased when they were added during the first hour of viral infection and remained when they were added after viral infection and for a continuous period of 48 h. Therefore, it was concluded that 2-SC 5 and 58 interfere more in the late stages of the virus life cycle that follow the enhancement of virus in cells. The obtained results showed that the type of substituent present at C-4′ on the B-ring influenced the potency of the anti-norovirus activity. As an example, this can be observed comparing the 4′-unsubstituted 2-SC 1 with 2-SC 21 (4′−OH substituted), 56 (4′−Cl substituted), 57 (4′−CH3 substituted), and 58 (4′−OCH3 substituted). The presence of −Cl or −OH at C-4′ on the B-ring led to a loss of the antiviral effect of 2-SC 56 or 21, respectively. 2-SC 57, with −CH3 at C-4′ on the B-ring, showed some antiviral activity but was approximately three times less potent than 2-SC 58, with −OCH3 at C-4′ on the B-ring. Thus, −OCH3 at C-4′ on the B-ring showed to be the best substituent, when there is no other substituent present in the 2-SC structure. In what concerns the C-5 substitutions on the A-ring, the presence of −OBn in 2-SC 81 led to a loss of antiviral effect, while the presence of −OH in 2-SC 5 led to one of the most active compounds. The existence of the −CH3 at C-α of the double bond of the styryl moiety led 2-SC 82 to show antiviral effect. The presence of the −OCH3 at C-3 on the C-ring appears to confer some antinorovirus activity to 2-SC 70 . In what concerns S. aureus, two strains were studied: ATCC 29212 and ATCC 43300. 2-SC 78 and 86–89 were effective against both strains for ATCC 43300 strain; however, 2-SC 92 was only effective against the ATCC 29212 strain (DIZ = 9 ± 0.12 mm). 2-SC 78 were effective against S. sciuri and S. xylosus , while 2-SC 90 was only effective against S. sciuri (DIZ = 12 ± 0.06 mm). In what concerns the Gram-negative bacteria, the studied 2-SC did not show any antibacterial activity against P. aeruginosa and K. pneumoniae. 2-SC 86–90 showed activity against E. coli strain ATCC 25922, and additionally, 2-SC 89 and 90 also showed activity against E. coli strain ATCC 35218. Analysing the structures of the tested 2-SC, in general, the 2-SC with −F on the A- and/or B-rings (78 and 86–91) were effective against a large number of studied bacterial organisms, which seems to indicate that −F plays an important role for the antibacterial activity. 2-SC 78, with only one −F at C-6 on the A-ring, showed an inhibitory effect against all the studied Gram-positive bacteria but did not show any activity against any of the Gram-negative bacteria. 2-SC 90, with −F at C-7 on the A-ring and C-4′ on the B-ring, showed antibacterial activity only against two Gram-positive bacteria (B. subtilis and S. sciuri) but was more effective than 2-SC 78 against these specific strains. Additionally, 90 also showed an inhibitory effect against both strains of E. coli (Gram-negative bacteria). 2-SC 89, with −F at C-3′ and C-5′ on the B-ring, also exhibited inhibitory effect against both strains of E. Coli and against B. subtilis and both strains of S. aureus. The presence of only one −F at C-2′ (86) or at C-3′ (87) or at C-4′ (88) on the B-ring led these 2-SC to be effective against B. subtilis and both strains of S. aureus and only one strain of E. coli (ATCC 25922). The obtained results in this study seem to indicate that the position and number of −F substituents in the A- or B-rings influence the antibacterial activity of 2-SC. 2-SC 58, 92, and 93 have no −F substituents in their structure. 2-SC 58, with −OCH3 at C-4′ on the B-ring, was only active against B. subtilis. The presence of −OCH3 at C-3′ and C-4′ on the B-ring (92) led to an inhibitory effect against B. subtilis and one strain of S. aureus (ATCC 29212). The 2-SC 93 is a methylenedioxy derivative and displayed no antibacterial activity, which apparently indicates that this group does not favour this activity .The existing works on the antibacterial activity of 2-SC include studies with different strains of Gram-positive and Gram-negative bacteria. The antibacterial activity demonstrated by 2-SC showed some variability, and it is not possible to define a clear SAR for each strain, resulting in the establishment of a generic SAR for the antibacterial activity. Thus, the above-mentioned structural characteristics that demonstrated to favour the antibacterial activity of 2-SC are summarized in Candida albicans, Aspergillus niger, Aspergillus flavus, and Penicillium chrysogenum and are below discussed. All the observed antifungal effects are summarized in Antifungal agents are used to treat fungal infections, in which the most common types are cutaneous, and ringworm infections of the skin and nails , 50. Ano94–101 , A. niger (NCIM-1196), and A. flavus (NCIM-539) by the agar dilution method. In this study, the results were presented as MIC values but the associated error was not indicated, even though the assays were performed in triplicate. 2-SC 94 and 96 were the most potent (MIC = 25 μg/mL) against C. albicans, being two times more potent that positive control, fluconazole (MIC = 50 μg/mL). For the A. niger, 2-SC 94 and 96–98 were the most potent (MIC = 25 μg/mL) and were equipotent with fluconazole. Against A. flavus, 2-SC 94, 96, and 98 were the most potent (MIC = 25 μg/mL), being equipotent with fluconazole. Analysing the substitution pattern, all the tested 2-SC have a 1,2,4-triazole ring at C-4′ on the B-ring. Interestingly, 2-SC 94, with only a 1,2,4-triazole ring at C-4′ on the B-ring as a substituent, showed an excellent antifungal activity against all the tested fungal strains. The presence of only one −CH3 at C-6 on the A-ring (95) led to a decrease in antifungal activity, when compared to 2-SC 94 and 96. The presence of a −Cl or −Br at C-6 on the A-ring  also led a decrease in antifungal potential compared to 2-SC 94. The existence of a −Cl at C-6 and C-8 on the A-ring (100) also did not favour antifungal activity. The introduction of −F at C-6 on the A-ring (98) maintained the antifungal activity against A. niger and A. flavus but decreased the potential against C. albicans, when compared to 2-SC 94. The studied 2-SC were also evaluated for their ADMET, as already mentioned in α-demethylase. The 1,2,4-triazole ring at C-4′ on the B-ring was mainly responsible for the interaction in the active site. Overall, a good binding interaction was shown between 2-SC 94–101 and the active site of fungal enzyme cytochrome P450 lanosterol 14 α-demethylase . Interestingly, 2-SC 58 was five to six times less potent at inhibiting the growth of MRC-5 cells (GI50 = 26.9 ± 3.2 μM). Regarding proliferation of peripheral human lymphocytes, 2-SC 58 (9.4–600 μM) showed to be a weak inhibitor (IC50 = 74.7 ± 4.8 μM). The cell cycle of non-tumor MRC-5 cells does not appear to be affected by 2-SC 58, since the cells presented a profile of DNA content similar to the untreated control. On the other hand, 2-SC 58 led to the hold in the G2/M phase of the cell cycle of MCF-7 cells in a concentration- and time-dependent manner. Also, for NCI-H460 cells, the effects of 2-SC 58 on the cell cycle were concentration and time dependent and led to an arrest at the G2/M phase of the cell cycle. These results led the authors to analyse the effect of 2-SC 58 (2.2–180 μM) on confluent (non-proliferative) and non-confluent  cells with the assumption that 2-SC 58 specifically affects mitosis. The results of both cell lines showed a high cellular protein content for the confluent cells, indicating that they were less sensitive to the 2-SC 58 treatment than non-confluent cells. The evaluation of cell growth by DNA synthesis for the NCI-H460 cell line indicates an inhibition of cell growth in non-confluent cells, in a concentration-dependent manner. The evaluation of the effects of 2-SC 58 (100 μM) on cytoskeleton components (actin filaments and microtubules) of MCF-7 and NCI-H460 cells during the interphase showed that the microtubule cytoskeleton appeared rich and intact in both cell lines, similar to the untreated cells of the control. Mitotic cells have undergone serious alterations, such as monopolar microtubule arrays, and rarely, normal mitotic spindles were found. For actin filaments, no significant changes were detected in both cell lines after 2-SC 58 treatment. These results suggest that 2-SC 58 specifically affects microtubules during mitosis. In the in vitro tubulin polymerization assay, 2-SC 58 (100–500 μM) led to a slight increase in tubulin polymerization, in a concentration-dependent manner, and the highest concentration tested (500 μM) resulted in the maximum polymerization plateau. In conclusion, 2-SC 58 appears to be a potential microtubule-stabilizing agent and a selective proliferation inhibitor of human tumor cell lines over normal cells, acting preferentially on proliferating cells , except for 2-SC 116, 119, 122, and 127 that were not active. Finally, 2-SC 121, 125, and 131 demonstrated good antiproliferative activity against BT483 cells, with IC50 values of 16.6 ± 2.4 μM, 15.5 ± 1.9 μM, and 16.6 ± 1.3 μM, respectively. The HeLa cell line was the most sensitive to the tested 2-SC, since most the tested 2-SC had better antiproliferative effects against this cell line when compared to the other tested cell lines, only 2-SC 119 and 124 were not active. 2-SC 129 was the most cytotoxic against the HeLa cell line (IC50 = of 4.9 ± 1.0 μM). The results obtained for the SKHep cell line showed that 2-SC 119, 120, 125, 127, and 128 were not active and 2-SC 124 and 129 were the most potent . The results of 2-SC 129 obtained for the HeLa cell line led the authors to make a mechanistic study of its antiproliferative effects. Thus, HeLa cells were treated with several concentrations of 2-SC 129 (1–10 μM), and the results showed that suppression of cell proliferation was concentration dependent. The effects of 2-SC 129 (5–20 μM) on HeLa cell morphology were also studied, and significant morphological changes have occurred, such as cell rounding and detachment from the substratum. The effects of 2-SC 116, 122, and 129 on apoptosis and cell cycle profile of HeLa cells were also evaluated to establish a relationship between the antiproliferative effect and cell cycle arrest. 2-SC 116 and 122 (20 μM) led to some cell cycle arrest in the subG1 phase, while 2-SC 129 (5–20 μM) induced cell cycle arrest in the subG1 phase in a concentration-dependent manner, which may indicate that the antiproliferative effect of 2-SC 129 observed in HeLa cells may be due to DNA fragmentation, resulting in cell death. Therefore, the effect of 2-SC 129 (5 and 10 μM) on DNA fragmentation in HeLa cells was also studied, by 4′,6-diamidino-2-phenylindole (DAPI) staining, and this fragmentation clearly occurred after treatment with 10 μM of 2-SC 129. The possibility of 2-SC 129 (10 μM) to have a microtubule-stabilizing effect was also evaluated by in vitro microtubule polymerization assay, and no effect was observed, suggesting that the antiproliferative effect on HeLa cells occurs by the other mechanisms mentioned above. In this study, the 3D-QSAR model was established by comparative molecular field analysis (CoMFA) on the BT483 cell line, to investigate how the 2-SC substituents influence the activities of the BT483 cell line. For this analysis, it was assumed that each 2-SC would adopt a conformation in its lowest energy, since the binding site is unknown. The results of the predictive model were in line with those obtained in cellular model. In what concerns the SAR, in this study, it is difficult to establish a relationship between the antiproliferative effects against the studied cell lines and the structures of the studied 2-SC, since the observed cytotoxic effects varied considerably among cell lines and regardless of the common structural characteristics among the studied 2-SC [Shaw and co-workers  synthesi116–131,  and evalied 2-SC .1, 56, 58, 59, 92, 93, 103, 104, 125, and 132–147 , HeLa, ovarian carcinoma (OVCA), SKHep, and large lung carcinoma (H460) was assessed. In addition, for 2-SC 103, growth inhibition against BT483 and colorectal adenocarcinoma (SW620) cell lines was also studied. For 2-SC 138–147, growth inhibition against AGS, PC-3, H460, and SW620 cell lines was studied. 2-SC 138 was the most active growth inhibitor (GI50 = 1.3 ± 0.07 μM) of AGS cells, followed by 2-SC 103 (GI50 = 2 ± 0.2 μM). 2-SC 1, 56, 92, 93, 104, 125, 132–134, and 143–147 did not inhibit AGS cell growth. Results of growth inhibition of 2-SC against HeLa cells showed that 2-SC 58, 59, 103, and 136 were the most potent, with GI50 varying from 6 ± 1 (103) to 9.9 ± 1 μM (136). 2-SC 56, 92, 132, and 134 did not inhibit HeLa cell growth. Against OVCA cells, 2-SC 56, 58, 103, and 135 were the most active, with GI50 values between 6 ± 1 (58) and 11 ± 1 μM (56), and 59, 93, 125, 132, 134, and 137 did not inhibit OVCA cell growth. 2-SC 58 was the most potent growth inhibitor (GI50 = 5 ± 1 μM) against the SKHep cells, and 1, 92, 93, 125, 132, and 135 did not inhibit SKHep cell growth. Results of growth inhibition of 2-SC against H460 cells showed that 2-SC 58, 103, 135, 139, and 141 presented the lowest GI50 values, varying from 6 ± 1 (103) to 10 ± 2 μM (139 and 141), and 1, 92, 93, 132, 138, 140, and 142–147 did not inhibit their growth. Only 2-SC 138 and 139 were able to inhibit the growth of PC-3 cells . For SW620 cells, 2-SC 138, 139, and 141 were able to inhibit their growth, while 2-SC 140 and 142–147 did not. Only 2-SC 103 was tested against BT483 cells and a GI50 value of 10 ± 2 μM was found. Doxorubicin was tested as a positive control against all carcinoma cell lines and showed GI50 values between 0.02 ± 0.01 and 1.76 ± 0.71 μM. The results obtained in this study reinforces the idea, previously mentioned [3 on the B-ring is related to the antitumoral activity. The results obtained for 2-SC 58 show that an −OCH3 at C-4′ on the B-ring is favourable for the inhibition of growth against the studied carcinoma cell lines, with GI50 values being relatively low. 2-SC 58 was one of the 2-SC with the best activity. 2-SC 103 that has −OCH3 at C-3′, C-4′ and C-5′ on the B-ring also presented good activity. Thus, the position that seems to most favour the activity is −OCH3 at C-4′ on the B-ring (58) or simultaneously at C-3′, C-4′, and C-5′ on the B-ring (103). In addition to −OCH3, the tested 2-SC also displayed other groups as substituents on the B-ring. Interestingly, different halogen substituents resulted in different activities: 2-SC 104, with −Br at C-4′ on the B-ring, showed growth inhibition activity against HeLa, OVCA, SKHep, and H460 cells; 2-SC 136, with −F at C-3′ and C-4′ on the B-ring, showed activity against all the studied carcinoma cells; 2-SC 56, with −Cl at C-4′ on the B-ring, demonstrated activity against OVCA, SKHep, and H460 cells. The introduction of −CF3 into C-4′ on the B-ring led 2-SC 137 to display activity against AGS, HeLa, SKHep, and H460 cells. In turn, the introduction of an −OCF3 into C-4′ on the B-ring led 2-SC 125 to only display activity against HeLa and H460 cells. 2-SC 59 has a −NO2 at C-4′ on the B-ring and was only inactive against OVCA cells and showed better activity than 2-SC 125 and 137. Since the 2-SC 103 demonstrated good activity, analogues were synthesized with −OCH3 at C-3′, C-4′ and C-5′ on the B-ring but with modifications on the A-ring (138–142). In general, the analogues (138–142) were less active than 2-SC 103, except for 2-SC 138 against AGS cells, which showed great potency. Another series of 2-SC (143–147) was also synthesized, but now with −OH at C-3′, C-4′, and C-5′ on the B-ring and modifications in several positions on the A-ring. However, none of these 2-SC showed growth inhibitory activity against the studied carcinoma cell lines, which seems to indicate that the simultaneous presence of −OH groups on the A- and B-rings does not favour this activity. The importance of double bond of the styryl moiety and the rigidity of the 2-SC structure for growth inhibitory activity was also evaluated. For this, a structure analogous to 2-SC 103 was tested, in which the double bond of the styryl moiety was hydrogenated to a single bond. This analogue and 2-SC 103 were tested against seven carcinoma cell lines . The new analogue showed a decreased activity when compared to 2-SC 103. Thus, the double bond of the styryl moiety seems to favour the growth inhibitory activity [For 2-SC entioned , 66, thaactivity .7–11  was assayed by the MTT assay, and taxol was used as positive control. In general, 2-SC 7–11 showed to be more effective against HepG2 and KB cells, being more cytotoxic for these two cell lines than for MDA-MB-231 and SMMC-7721. 2-SC 7 was the most cytotoxic against HepG2 cells (IC50 = 6.8 ± 0.3 μM), followed by 2-SC 8 (IC50 = 7.4 ± 0.1 μM). 2-SC 8 showed to be the most potent against MDA-MD-231 cells (IC50 = 8.7 ± 0.3 μM), followed by 2-SC 7 (IC50 = 11.9 ± 0.6 μM). 2-SC 9 was the most cytotoxic against KB cells (IC50 = 3.0 ± 0.2 μM) and SMMC-7721 (IC50 = 14.6 ± 0.3), and 11 was considered inactive in the last ones. Taxol was tested against the four cancer cell lines and showed IC50 values that ranged from 0.04 ± 0.002 to 1.1 ± 0.2 μM. These 2-SC have in common an −OH at C-5 and C-7 on the A-ring and at C-4′ on the B-ring, and their structure differ in the substituents present at C-6 and C-8 on the A-ring. The −CH2CH=C(CH3)2 group seems to contribute to the greater cytotoxic effect observed against HepG2 and MDA-MB-231 cells (7 and 8), while the −CH2CH(OH)CH=CH2 group seems to favour cytotoxicity against SMMC-7721 and KB cells (9 and 10). In conclusion, the presence of substituents at C-6 and C-8 on the A-ring (7–10) seems to favour the cytotoxic activity over the unsubstituted structure (11) at these positions [Yang and co-workers  isolated7–11  and repo148–151, 148 was the most cytotoxic against A549 cells (IC50 = 17.9 ± 2.9 μg/mL). 2-SC 151 showed potent cytotoxicity against HCT-15 (IC50 = 9.22 ± 1.8 μg/mL). Only 2-SC 148 showed cytotoxicity against KB cells (IC50 = 34.98 ± 6.6 μg/mL). None of the 2-SC 148–151 showed cytotoxic activity against SK-MEL-2 cells, up to a concentration of 100 μg/mL. The positive control, cisplatin, was tested against four cancer cell lines and showed IC50 values that ranged from 0.56 ± 0.12 to 1.65 ± 0.12 μg/mL. 2-SC 148–151 have in common an −OH at C-7 on the A-ring and a substituent structurally derived from lavendustin A at C-8 on the A-ring, and differ in the substituents on the B-ring. 2-SC 148 that has no substituent on the B-ring exhibited cytotoxicity against A549, HCT-15, and KB cells. In turn, 2-SC 149 that has an −OCH3 at C-4′ on the B-ring showed a decrease in the cytotoxic effect when compared to 2-SC 148, as it only showed effect against A549 and HCT-15 cells and with higher IC50 values. 2-SC 150, with −OCH3 at C-3′ and C-4′ on the B-ring, also revealed a decrease in the cytotoxic effect when compared with the activity of 2-SC 148. The presence of −Br at C-3′ on the B-ring (151) seems to favour the cytotoxic effect against HCT-15 cells, but to decrease the activity against A549 cells, when compared to 2-SC 148. In general, in the presence of a substituent structurally derived from lavendustin A at C-8 on the A-ring, the modifications at C-3′ and/or C-4′ on the B-ring did not favour the cytotoxic activity [Lee and co-workers  synthesiactivity .152–154  [152–154 (1.56–100 μM) to activate the Keap1-Nrf2 pathway was evaluated and compared to xanthohumol and sulforaphane, which were tested as positive controls, in AREc32 cells. The results were presented as the concentration required to increase luciferase activity by fivefold in relation to solvent control , but the authors did not mention the associated error. 2-SC 152–154 presented a concentration-dependent activity and showed C5 values of 2.9 μM, 18 μM, and 10.2 μM, respectively. The xanthohumol and sulforaphane presented C5 values of 7.8 μM and 4.8 μM, respectively. Thus, 2-SC 152 had better Nrf2-inducing potential than the two positive controls. 2-SC 152–154 did not show cytotoxic effects against AREc32 cells. 2-SC 152 was able to reduce the viability and proliferation , while 153 did not affect cell proliferation or induce the death of K562 cells. When the effect of 2-SC 152 on the viability of peripheral blood mononuclear cells from healthy donors was evaluated, this compound did not affect the viability of these cells, which may indicate selective toxicity for cancer cells. The obtained results revealed that the introduction of −OCH3 at C-3′ and C-4′ (153) or only at C-4′ (154) on the B-ring reduced the Nrf2-inducing activity, when compared with 2-SC 152. The same conclusion can be drawn for the antiproliferative activity against the K562 cell line, since 2-SC 153 did not have an antiproliferative effect and 2-SC 152 did. In this study, the antioxidant activity of 2-SC 152–154 was also evaluated, using the ferric-reducing antioxidant power and DPPH methods. However, these 2-SC demonstrated very low or non-existent activity, therefore this study was not mentioned in the Talhi and co-workers  evaluate152–154 . These as (K562) . The Nrfs (K562) , 72. The155–163 , melanoma, ovarian, renal, prostate, and breast. In this study, the error associated with the results was not indicated. In the first screening, 2-SC 160 showed growth inhibitory activity against four leukaemia cell lines , two non-small cell lung cancer cell lines , one CNS cancer cell line (% GI = 67%), two ovarian cancer cell lines , three renal cancer cell lines , one prostate cancer cell line (% GI = 72%), and one breast cancer cell line (% GI = 86%). 2-SC 163 inhibited the growth of five leukaemia cell lines , two non-small cell lung cancer cell lines , four colon cancer cell lines , two CNS cancer cell lines , three melanoma cell lines , four ovarian cancer cell lines , three renal cancer cell lines , two prostate cancer cell lines , and one breast cancer cell line (% GI = 70%). After this primary screening, 2-SC 160 and 163 (0.01–100 μM) were selected by the authors for an assay against cell lines of the nine types of cancer mentioned above. In this assay, GI50, the compound concentration resulting in total growth inhibition (TGI), and LC50  were calculated for each cell line. Summarizing and in general, 2-SC 163 (median GI50 = 1.9 μM) was more active than 160 (median GI50 = 2.45 μM) against the tested cell lines. The structures of these two 2-SC differ in the substituent present at C-4′ on the B-ring: 160 has a −Cl, while 163 has −N(CH3)2. These results indicate that the −N(CH3)2 at C-4′ on the B-ring favours the growth inhibitory activity in relation to the −Cl in the same position. After the results are obtained in the screening, the authors submitted the 2-SC 160 and 163 to the kinase inhibition assay to determine whether the activity of these 2-SC was related to their enzyme inhibition ability of protein kinase targets. Thus, the profile of 2-SC 160 and 163 against cyclin-dependent kinase (CDK) 2/cyclin E1, CDK4/cyclin D1, and glycogen synthase kinase 3-beta (GSK-3β) was performed [β is an enzyme that regulates numerous cellular signalling pathways and is involved in a wide range of cellular processes, such as cell cycle regulation and proliferation [160 and 163 did not inhibit the enzyme activity. 2-SC 160 and 163 showed weak inhibition against the CDK4/cyclin D protein kinase target but caused stimulation of GSK-3β activity  instead of inhibiting it. Given that 2-SC 163 was very potent in cell growth inhibition assays, it was not expected to stimulate of GSK-3β activity, but to inhibit it. Thus, this result led the authors to progress to in vivo studies with 2-SC 163 against breast cancer induced in female BALB/mice (Ehrlich solid tumor), in an attempt to correlate the high in vitro antiproliferative activity with the stimulation of GSK-3β activity. The tumor was induced and grew for two weeks, after which the mice received intraperitoneal injections of 2-SC 163 (3 mg/kg and 6 mg/kg) for five days, daily. The treatment with 2-SC 163 inhibited the growth of the tumor in a dose-dependent manner. The treatment with 2-SC 163 at low and high dose  significantly decreased the level of cyclin D1 in the tumor, whereas it increased the level of GSK-3β, when compared to the untreated group. Thus, the 2-SC 163 antitumor effect was accompanied by a significant decrease in cyclin D1 levels and a significant increase in GSK-3β levels. These results seem to indicate that tumor suppression is partially mediated by an increase in GSK-3β and a decrease in cyclin D1 levels, which suggests that cyclin D1 levels can be reduced by the activation of GSK-3β that phosphorylates cyclin D1, leading to its proteolysis [Ragab and co-workers  syntheti155–163 , with a erformed . CDKs plferation , 75. Proteolysis .164  decreased the mitochondrial membrane potential, which was more pronounced at the highest concentration. 2-SC 164 showed an inhibitory effect, in a concentration-dependent manner, on respiratory rates. Thus, the decrease in transmembrane potential induced by 164 can result from the inhibition in any respiratory complex , since it must interact with the phosphorylation system and the mitochondrial redox chain. The authors, in an attempt to elucidate the exact action of 2-SC 164 in the respiratory redox chain and in the phosphorylation system (directly on the respiratory complexes or by an uncoupler effect), evaluated the effect of 2-SC 164 on the respiratory complexes  and in the mitochondria swelling. 2-SC 164 decreased the ATP synthase and ATPase activities  and inhibited succinate dehydrogenase activity (17%). 2-SC 164 partially inhibited cytochrome c oxidase (27%), however it inhibited succinate cytochrome c reductase (≈62%). These results may indicate that 2-SC 164 essentially interacts with electron transfer at the level of complex III. Experiments in the osmotic potassium acetate medium showed that in the presence of 2-SC 164, only a small mitochondrial swelling was observed, in a concentration-dependent manner, when compared to carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, used as a positive control. In conclusion, the results of this study indicate that the possible toxicological effect of 2-SC 164 may be related to the inhibition of the phosphorylation system and the electron transfer via the respiratory chain of rat liver mitochondria, especially in complex III (succinate cytochrome c reductase). The authors also suggested that 2-SC 164 may induce apoptosis through a decrease in the cellular energetic charge and promotion of the release of cytochrome c in the cytosol. Thus, the induction of mitochondrial cytochrome c release, shown in these biochemical studies, can subsequently trigger an apoptotic mechanism, an indicator of the potential cancer chemopreventive activity of 2-SC 164 [Peixoto and co-workers  studied 164  on rat l2-SC 164 .The great number of studies found in the literature about 2-SC antitumoral activity covers a wide variety of tumor cell lines, some also include normal cell lines, but not all the studies. Moreover, they comprise a wide range of 2-SC with numerous substituents in different locations, making it difficult to indicate which type of cancer is most studied and to establish a SAR. However, the above-mentioned structural characteristics that demonstrated to favour the antitumoral activity of 2-SC are summarized in 34–39, and 42–50, 50 and SI were calculated and presented. The authors mentioned the obtained CC50 values for each cell line, without the associated error, and the mean of the CC50 values obtained for the four tumor cell lines and the three normal cells, with the associated error. 3-SC 46 was the most cytotoxic against the four tumor cell lines (mean CC50 = 2.0 ± 1.2 μM), followed by 37 (mean CC50 = 6.4 ± 2.4 μM). Against normal oral cells, 3-SC 46 and 37 showed lower cytotoxicity . 3-SC 39 was also quite cytotoxic against tumor cell lines (mean CC50 = 13 ± 6.1 μM) and showed lower cytotoxicity against normal cell lines (mean CC50 = 339 ± 183 μM). Thus, these 3-SC showed high tumor specificity  and their results can be compared to those obtained for the positive controls, doxorubicin and 5-fluorouracil . Only 3-SC 48 did not show cytotoxicity against tumor cell lines nor against normal oral cell lines. In what concerns QSAR analysis, several chemical descriptors were used. The effects of functional groups on cytotoxicity of the studied 3-SC against tumor cell lines were evaluated, and the substituents −OCH3 at C-6 on the A-ring and −OH at C-4′ on the B-ring were defined to be crucial to increase the cytotoxicity against tumor cell lines. Accordingly, 3-SC 37 that has −OCH3 at C-6 on the A-ring and at C-4′ on the B-ring showed a greater cytotoxicity and tumor specificity than 3-SC 39, with −OCH3 at C-6 on the A-ring and at C-3′, C-4′ and C-5′ on the B-ring. 3-SC 38 and 39 have −OCH3 at C-6 on the A-ring and at C-3′ and C-4′ on the B-ring, but 39 also have an −OCH3 at C-5′ on the B-ring and showed better tumor specificity than 38, which indicates that this additional −OCH3 may contribute to the cytotoxic activity when in the presence of multiple −OCH3 groups on the B-ring. 3-SC 34 (with −OCH3 at C-4′ on the B-ring), 35 (with −OCH3 at C-3′ and C-4′ on the B-ring), and 36  showed low tumor specificity, which seems to indicate that the presence of −OCH3 groups only on the B-ring (without substituents at C-6 on the A-ring) decreases the cytotoxic activity against tumor cell lines. When an −OCH3 at C-6 on the A-ring is present, −Cl at C-4′ on the B-ring (43) seems to favour the cytotoxic activity against tumor cell lines over the presence of −F in the same position (42). 3-SC 44 (with −OH at C-4′ on the B-ring) and 45 (with −OH at C-3′ and C-4′ on the B-ring) have no substituents in the A-ring and showed better tumor specificity than 3-SC also without substituents in the A-ring but with −OCH3 on the B-ring, in the same positions (34–36), which may indicate that −OH on the B-ring is important for the cytotoxic activity against tumor cell lines. In 3-SC 46, −OCH3 is at C-6 on the A-ring and −OH is at C-4′ on the B-ring; while in 3-SC 48, −OCH3 is at C-4′ on the B-ring and −OH is at C-6 on the A-ring; this inversion of the substituents' positions led to the loss of 3-SC 48 cytotoxic activity. As mentioned, 3-SC 46 has an −OCH3 at C-6 on the A-ring and −OH at C-4′ on the B-ring and it showed better tumor specificity than 47, which has an additional −OH at C-3′ on the B-ring. Moreover, 3-SC 49 and 50 both have −OH at C-6 on the A-ring and C-4′ on the B-ring but 50 has an additional −OH at C-3′ on the B-ring that led to a better tumor specificity than 49. This result indicates that when C-6 on the A-ring is −OH instead of −OCH3, the addition of −OH to the B-ring can improve tumor specificity. In conclusion, 3-SC 46, with −OCH3 at C-6 on the A-ring and −OH at C-4′ on the B-ring, was the most cytotoxic and showed high tumor specificity, which indicates that these two groups, at these positions, favour the cytotoxic activity against the studied tumor cell lines [Shimada and co-workers  synthetie B-ring  seems to37, 39, and 46  than against epithelial malignant cell lines  [37, 39, and 46 demonstrated greater tumor specificity than the positive controls [46 showed high potency-selectivity to malignant tumors . To determine the minimum treatment time required for cytotoxicity induction, pulse-chase experiments were performed, where HSC-2 cells were incubated until 47 h with increasing concentrations of 37, 39, and 46 (0–1000 μM), and the relative viable cell number was determined by the MTT assay. 3-SC 46 was the most cytotoxic , followed by 37  and 39 . In this assay, it was also observed that high concentrations (100–1000 μM) of 3-SC 37 and 46 led to a cell death greater than 90%, while 3-SC 39, for the same range of concentrations, was less cytotoxic, showing a 30% cell viability. The obtained results led the authors to conclude that 3-SC 46 had the highest cytotoxicity and tumor specificity among the three 3-SC , with cytotoxicity being triggered at early 3 h after treatment. Therefore, the effects of 3-SC 46 on fine cell structures and the type of cell death were evaluated, as well as the metabolic profile induced by this 3-SC in HSC-2 cells. 3-SC 46 (3 and 10 μM) induced concentration-dependent mitochondrial vacuolization, which suggests induction of mitochondrial injury. 3-SC 46 (10 μM) suppressed autophagy and induced apoptotic cell death. In what concerns metabolomic analysis, 3-SC 46  did not significantly affect major cellular metabolic pathways, however the authors reported changes in metabolites involved in amino acid and glycerophospholipid metabolism [As described, 3-SC , and 46  showed tus study , the cytus study , the aut 6.1 μM) . Doxorub2, 40, 41, and 165–175, μM) was assessed by the MTT assay, and CC50, SI, and PSE were calculated and presented. In this study, the associated error to CC50 values was not indicated, even though the determination of the relative viable cell number was performed in triplicate. The CC50 values were presented for each cell line and the mean of the CC50 values of the four tumor cell lines, and the three normal cells were also presented, with the associated error. Doxorubicin (0.078–10 μM) and 5-fluorouracil (7.8–1000 μM) were used as positive controls. 3-SC 168 and 175 were the most cytotoxic against the tumor cell lines , followed by 169 (mean CC50 = 1.1 ± 0.31 μM). Against normal oral cells, 3-SC 168 and 175 showed low cytotoxicity , while 3-SC 169 showed high toxicity . Thus, 3-SC 168 and 175 showed high tumor specificity , being better than doxorubicin and 5-fluorouracil ; and 169 showed low tumor specificity (SI = 3.7). 3-SC 41 and 167 were also quite cytotoxic against tumor cell lines , but nevertheless also showed high cytotoxicity against normal oral cell lines . 3-SC 168 showed a high potency-selectivity expression (PSE = 49842.4), followed by 175 (PSE = 27898.1), being these values higher than those obtained for the positive controls (PSE = 24953.8 for doxorubicin and PSE = 26.2 for 5-fluorouracil). These results indicate that all tumor oral cell lines tested were more sensitive to 3-SC 168 and 175 than normal oral cells. Thus, the effects of 3-SC 168 (1–4 μM) and 175 (2–8 μM) on the apoptosis induction and growth arrest were further monitored by cell cycle analysis of HSC-2 cells. 3-SC 168 and 175 and the positive control, actinomycin D (1 μM), led to an accumulation of cell population in the subG1; however, only 3-SC 168 and 175 induced the accumulation at G2/M phase cells and induced mitotic arrest. Thus, the results demonstrated that these two 3-SC induced the apoptosis and mitotic arrest in HSC-2 cell more potently than actinomycin D. The QSAR analysis of all the tested 3-SC indicated that tumor specificity was correlated with the following descriptors: topological and 3D shape, size, electric state, aromaticity index, energy of the lowest unoccupied molecular orbital (LUMO), and partial charges. The authors also indicated that the presence of −OCH3 at C-6 on the A-ring reduced the tumor specificity and the presence of −OCH3 at C-7 on the A-ring led to an increase in tumor specificity. In fact, the 3-SC with the highest tumor specificity (168) has only −OCH3 at C-7 on the A-ring. Furthermore, the fact that 3-SC 166, with only an −OCH3 at C-6 on the A-ring, has a very low tumor specificity is an example that demonstrates that in addition to the type of substituents, their position also influences the tumor specificity. In general, the introduction of halogen atoms (−F and −Cl) at C-4′ on the B-ring (3-SC 40 and 41) reduced the tumor specificity, even in the presence of additional −OCH3 at C-7 on the A-ring (169 and 170). Unsubstituted 3-SC 2 also showed low tumor specificity. For 3-SC 165, 167, and 171, a low tumor specificity was found. These three 3-SC have in common the −N(CH3)2 group at C-4′ on the B-ring, which does not seem to favour tumor specificity. In conclusion, this study suggests that the −OCH3 group at C-7 on the A-ring favours the cytotoxic activity against the studied tumor cell lines and improves the tumor specificity of 3-SC [Takao and co-workers  synthetibicin 0.0–10 μM anAs for 2-SC, studies found in the literature on the antitumor activity of 3-SC include different tumor cell lines and a wide range of 3-SC with various substituents in different locations, making it difficult to establish a clear SAR. However, the above-mentioned structural characteristics that demonstrated to favour the antitumoral activity of 3-SC are summarized in 3 adenosine receptors, neuroprotective activity, and α-glucosidase inhibitory activity and are below discussed.In addition to the biological activities already described in the previous topics of this review, several SC have shown other less explored biological properties, such as affinity and selectivity for A3 adenosine receptors have only been reported in one study, to the best of our knowledge, in which three 2-SC were studied. Adenosine receptors are important targets for drug development as they have been associated with several biological functions, such as immune regulation and vascular function, but also with pathological processes, such as inflammatory and neurodegenerative diseases. Karton and co-workers [176–178 , acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). Years later, Takao and co-workers [1, 30, 56, 58, 88, 92, and 104–115, The potential of 2-SC as neuroprotective agents has only been studied in four studies, to the best of our knowledge. Yoon and co-workers  evaluate5  against 5  on the B-ring was also confirmed, because when they were methylated, the scavenging activity of 2-SC decreased. In contrast, the presence of −OCH3 on the B-ring (C-2′ or C-4′) seems to increase it. The presence of small alkyl groups at C-3 on the C-ring also seems to contribute to the antiallergic activity of the studied 2-SC.For the antiallergic activity, the presence of only one −CH3 at C-3 on the C-ring, and −NO2 at C-4′ on the B-ring seems to favour 2-SC antiviral activity against serotype 1B of HRV. These substituents also seem to be important for the antiviral activity of 2-SC against serotype 14 of HRV; however, the presence of −F at C-6 on the A-ring and −Cl at C-4′ on the B-ring also seems to contribute to this activity. 2-SC antiviral activity against MNV was also studied and demonstrated the importance of the presence of the −OH group at C-5 on the A-ring and the −OCH3 group at C-4′ on the B-ring for the antiviral activity against MNV. The inhibitory activity of 2-SC against the HIV-1 integrase was another of the studied antiviral effects, but only one 2-SC was evaluated, which was not very effective against the HIV-1 integrase. In what concerns 3-SC antiviral activity, there are few studies related to their antiviral properties, however in the reports found in the literature, 3-SC have shown few antiviral effects. 3-SC were studied against HIV, EV, and the serotypes 1B and 14 of HRV. The studied 3-SC did not show anti-HIV activity. In addition, it was only possible to conclude that the presence of −Cl at C-6 on the A-ring and C-4′ on the B-ring seems to play an important role for the antiviral effect against HRV 1B. The studied 3-SC showed low efficacy against EV and HRV 14, so it was not possible to establish a SAR.The antiviral potential of SC has been studied for different viruses, and the same SC have demonstrated different interactions with the different studied viruses and viruses' strains. These differences between the antiviral effects shown by SC indicate some selectivity, being an advantage for their use as antiviral agents. The analysis of the antiviral activity of 2-SC showed that it is significantly influenced by the substituents present in the structure and its location, however it is difficult to indicate in a generic way which substituents play the most important role. The presence of −Cl at C-6 on the A-ring, −OH or −OCH3 group at C-4′ on the B-ring also seems important for this activity. Summarizing, in part, the substituents that favour antibacterial activity also favour antifungal activity, which seems to indicate a weak selectivity by 2-SC.As previously mentioned, for the antibacterial and antifungal activities of SC, only 2-SC were studied and there are no reports of 3-SC with antibacterial and antifungal potential, to the best of our knowledge. The antibacterial activity of 2-SC was studied for different strains of Gram-positive and Gram-negative bacteria, with the tested 2-SC showing some variability in what concerns the results observed for each strain and between Gram-positive and negative bacteria, which makes it difficult to establish a clear SAR. Additionally, the studied 2-SC included several substituents which did not present coherent activities patterns, which also makes it difficult to establish of a specific SAR. Thus, only a generic SAR has been established for the antibacterial activity. The antibacterial activity of 2-SC seems to be influenced by the position and the number of −F substituents present on the A- or B-rings, as well as by the existence of −Cl or −OH at C-4′ on the B-ring. The C-6 on the A-ring and C-4′ on the B-ring also seem to be important substituents' positions for the antibacterial effects of 2-SC. For most 2-SC, the authors who reported their antibacterial properties, also assessed their antifungal potential. As reported for the antibacterial activity, the 2-SC studied included different substitution patterns. This fact, combined with differences in the used methodology between studies, only allowed the establishment of a generic SAR for antifungal activity. Similar to what was reported for the bacterial activity, the presence of the 1,2,4-triazole ring at C-4′ on the B-ring appears to contribute to the antifungal activity, as well as the existence of −Cl at C-4′ on the B-ring. The −OCHκB activation, and modulation of cytokines/chemokine production. Despite the reported studies being quite different and evaluating several mediators, the presence of −OH at C-3′ and C-4′ on the B-ring seems to generically favour the anti-inflammatory activity, similarly to what has been demonstrated for the antioxidant activity.Several anti-inflammatory mechanisms have been described for 2-SC, namely, inhibition of LFA-1/ICAM-1-mediated cell adhesion, interference with the arachidonic acid metabolic pathways, inhibition of NF-3 groups on the B-ring seems to play an important role in the antitumor activity performed by 2-SC, especially when the −OCH3 is at C-4′ on the B-ring. Furthermore, the simultaneous presence of −OCH3 groups at C-3′, C-4′, and C-5′ on the B-ring also seems to potentiate the antitumor effects of 2-SC. The styryl moiety seems to favour the growth inhibitory activity. The existence of −OCH3 at C-6 on the A-ring also appear to favour cytotoxic activity against tumor cell lines. All in all, the studies of the antitumoral activity of 3-SC seem to indicate that the presence of −OCH3 at C-6 or C-7 on the A-ring and a−OH at C-4′ on the B-ring favour cytotoxic activity against tumor cell lines.The antitumor potential is one of the most explored biological activities in the literature for SC. The antitumor properties of 2-SC are widely described, however there are few studies describing the antitumoral effects of 3-SC. Some of the existing studies, in addition to the evaluation of the effects of SC on tumor cell lines, also evaluate their effects on normal cells, which allows to understand whether the effects of SC are tumor selective. In addition to selectivity, it is important to know whether SC are also cytotoxic to normal cells. This fact is determinant for their use as possible antitumor agents, since the major objective is to demonstrate cytotoxicity against tumor cells, affecting as little as possible normal cells. Unfortunately, many of the studies reported did not include normal cell lines in their evaluation, making it difficult to understand if and in what extension SC are selective for tumor cell lines. In the cases where both cell types were studied, in general, SC were cytotoxic to tumor cells and did not show cytotoxicity to normal cells. These studies cover a wide variety of tumor cell lines, so it is difficult to indicate which type of cancer is most studied and to establish SAR. Nevertheless, in general, the presence of −OCHThe neuroprotective activity of SC is one of the biological activities less explored in the literature, since only the possible neuroprotective effects of three 2-SC were reported. Among these three 2-SC studied, only two showed neuroprotective properties: neuroprotective activity against glutamate-induced neurotoxicity in P12 pheochromocytoma cells and inhibition of AChE, BChE, and BACE1. Since the neuroprotective activity of 2-SC has been less studied and the effects evaluated are very different, it is difficult to identify the substituents that favour their effect, because in each study, a different 2-SC was evaluated.3 adenosine receptor, inhibitory activity of MAO-A and MAO-B, and inhibitory activity of α-glucosidase. In what concerns the affinity and selectivity of 2-SC for adenosine receptors, the presence of −OC2H5 at C-5 on the A-ring appears to favour the affinity and selectivity for the A3 adenosine receptor. For the MAO inhibitory activity, the presence of −OCH3 at C-6 on the A-ring and halogenated groups  at C-4′ on the B-ring favoured the MAO-A inhibitory activity; while the presence of −OCH3 at C-6 on the A-ring and −Cl or −Br at C-4′ on the B-ring favoured the MAO-B inhibitory activity and selectivity. The α-glucosidase inhibitory activity of 3-SC was influenced by the presence of the −OH group at C-6 on the A-ring and −OH at C-3′ and C-4′ or at C-4′ on the B-ring; however, the 3-SC with −OH at C-3′ and C-4′ on the B-ring was more potent than the 3-SC with just an −OH at C-4′ on the B-ring.As mentioned in the beginning of this section, in addition to the biological activities mentioned above, other activities have also been reported for SC, namely, affinity and selectivity for the AThis work demonstrates that SC exhibit a large variety of biological properties. Throughout this work, it can be observed that a wide variety of SC structures has already been studied, most of which with substituent variations on the A- and/or B-rings. The type of substituents in the SC structure and their location assume an essential role in each one of the activities shown. This variety, added to the experimental differences between studies, makes it difficult to establish a generic SAR for each activity. The antioxidant and antitumor activities of SC are undoubtedly the most explored ones in the literature. In conclusion, SC present a molecular scaffold that demonstrates an enormous potential for the development of new drugs envisioning the treatment of several oxidative process-related diseases, namely, inflammatory, carcinogenic, and neurodegenerative.


Expression and activation levels of 187 proteins, of which 137 were kinases and downstream substrates, were captured using the Reverse Phase Protein Array (RPPA). The DMSO effect was heterogeneous across cell lines and varied based on concentration, exposure time, and cell line. Of the 187 proteins measured, all were statistically different in at least one comparison at the highest DMSO concentration, followed by 99.5% and 98.9% at lower concentrations. Only 46% of the proteins were found to be statistically different in more than 5 cell lines, indicating heterogeneous response across models. These cell line specific alterations modulate response to in vitro drug screening. Ultra-low DMSO concentrations have broad and heterogeneous effects on targetable signaling proteins. Off-target effects need to be carefully evaluated in pre-clinical drug screening and testing.Targetable alterations in cancer offer novel opportunities to the drug discovery process. However, pre-clinical testing often requires solubilization of these drugs in cosolvents like dimethyl sulfoxide (DMSO). Using a panel of cell lines commonly used for in vitro drug screening and pre-clinical testing, we explored the DMSO off-target effects on functional signaling networks, drug targets, and downstream substrates. Eight Non-Small Cell Lung Cancer (NSCLC) cell lines were incubated with three concentrations of DMSO (0.0008%, 0.002%, and 0.004%  The identification of targetable alterations in cancer continues to offer novel opportunities to the drug discovery process and to the development of compounds capable of directly interacting with and modulating activation of malfunctioning proteins ,2. BecauWhile genomic alterations initiate and promote tumorigenesis, phenotypically these alterations manifest as aberrantly activated proteins and kinase-driven signal transduction events ,7. Withiv/v [v/v still modulate gene expression and large-scale miRNA profiles as well as tissue-specific genome-wide methylation patterns [To minimize DMSO-dependent cytotoxicity , its effv/v ,13. Howepatterns ,15. Thus−4 to 4 × 10−3 % v/v) on the signaling network of eight commercially available cell lines commonly used for in vitro drug testing and cell-based assays. Dose- and time-dependent changes in signaling events were captured on a continuous scale using the Reverse Phase Protein Microarray (RPPA), a high throughput antibody-based assay. To fully capture off-target effects and functional deregulation of signaling networks associated with DMSO, emphasis was placed on activation levels of kinases and downstream substrates commonly targeted by FDA approved and investigational anti-cancer compounds.Using lung cancer as a model system, this work explored the effect of ultra-low DMSO doses  cell lines was selected for molecular analysis. Cells were incubated with three different amounts of DMSO, namely 0.0008% (C1), 0.002% (C2), and 0.004% (C3) th rates . Cell lip = 0.01, 0.04, and 0.03 for C1, C2, and C3, respectively), while it promoted signal transduction events in others . The H2122 and H1734 lines were the most affected cell lines and presented a nearly 40% increase in the overall phosphorylation levels; however, the magnitude of change varied greatly across timepoints. On the other hand, the H358 and H23 cell lines were the least affected models within and across concentrations (p > 0.05). However, datapoints trended in similar directions across different concentrations of DMSO, suggesting that DMSO induces similar biological effects regardless of its amount.To explore the effect of DMSO on functional signaling networks and post-translationally modified proteins, broad changes in phosphorylation levels of 125 kinases and downstream substrates were first evaluated . Fold-chIn contrast, DMSO had an inhibitory effect on phosphorylation events in the H522 cells, especially 1 and 6 h after the administration of DMSO. Similarly, a 20% to almost 40% reduction in phosphorylation events was detected after 1 h of incubation in the H1838. The ability of DMSO to change signaling network activities, especially at earlier timepoints after incubation, may be of relevance when testing targeted compounds as these changes may alter a compound’s activity, its ability to engage with its target(s), and its effect on downstream substrates. To further explore the effect of DMSO on the overall signaling network, weighted Pearson’s correlation coefficients were calculated and their distribution was represented as histograms and kernel density plots . This apIn contrast, interconnection in the H358, H2122, and H522 never recovered baseline levels within the 24 h period . Howeverp < 0.05) were found in less than half of the measured analytes in C1 and C2 . However, the proportion of significant endpoints reached 63.6% at the highest DMSO concentration  were statistically different in at least one cell line for the DMSO concentration C1, while the number of statistically different endpoints increased to 186 (99.5%) in C2, and to 187 (100%) C3 . On a centration . Only 64ntration A. The H2ntration . A totalntration A. Of the 187 proteins evaluated, 86 (46.0%) were significantly different in more than 5 cell lines in C1 followed by 120 (64.2%) in C2 and C3 . AmongstIn the Calu-3 and H358 models, ERK 1/2 activation was significantly reduced throughout the incubation period, while in the H1734 line, ERK 1/2 activation levels progressively increased by 2 to 3 times and never recovered to baseline levels. Trends in ERK 1/2 activation in C2 and C3 followed activation patterns resembling those of C1 A.Activation patterns across time also varied from cell line to cell line for AKT, another key regulator of a complex signaling axis. DMSO induced downregulation of AKT S473 in six of the eight cell lines and only the H522 fully recovered after 24 h of incubation D. Increap < 0.05) across timepoints for all eight cell lines in C1, seven cell lines in C2, and five cell lines in C3 , we evaluated broad responses to targeted agents and standard chemotherapeutic compounds across our eight cell lines. Drug susceptibility information was collected on average for 180 compounds with the exception of the H1838 and H1734 . For each compound and cell line, the GDSC database provides IC50-based Z-scores. Cell lines with Z-scores > +2 are considered resistant to treatment while those with Z-scores < −2 were defined as sensitive. Of interest, H2122, the cell line most affected by DMSO in our analysis, showed the greatest sensitivity across all compounds , followed by H23 (2.8%) and Calu-3 (0.5%) , were amongst the models with the highest sensitivity to different anti-ERK compounds. The Calu-3 cells were the only exception to this pattern. Taken together, these data indicated that the DMSO effects on signaling events and drug targets is highly heterogeneous. Cell-dependent response to DMSO may differentially affect activation levels of drug targets and downstream substrates across cell lines and should be carefully evaluated in drug screening and in vitro testing.Finally, using the Genomics of Drug Sensitivity in Cancer (GDSC) database (3 (0.5%) A. The H23 (0.5%) B. In conTo our knowledge, this is the first report exploring broad off-target effects of DMSO on protein signaling networks with a focus on biochemically interconnected, post-translationally modified drug targets and downstream substrates. With a growing number of targeted compounds entering the market every year, discerning the effects of experimental variables from true drug-associated effects is of primary importance to improve the drug discovery process and pre-clinical testing. While DMSO remains the most commonly used cosolvent for water insoluble compounds in pharmacological and toxicological studies, the concentration at which it is used is often unreported . SubsequIt has been previously documented that DMSO is a non-inert substance with significant effect across macromolecules and subsequently cellular functions and molecular profiles ,20,21,22v/v ), selectively induces phosphorylation of tyrosine residues on the EGF receptor in crude membrane fractions of murine erythroleukemia cells [In vitro studies and biochemical assays have previously reported that exposure to DMSO can promote phosphorylation of kinases on multiple phospho-epitopes ,32,33. Eia cells . In lineActivation of ERK 1/2, a central node of the proliferative MAPK pathway and a crossroads between signal transduction networks, is a good example of these diverse adaptation mechanisms as each of the eight models analyzed in this work showed distinct patterns of ERK 1/2 activation and recovery after 24 h of incubation with DMSO. These trends appeared to be independent from expression and activation of cell cycle regulatory molecules or changes in ERK 1/2 overall expression levels, suggesting that DMSO directly affects ERK’s signaling activity. Similarly, Cataldi and colleagues have previously demonstrated that incubation with DMSO for 24 h induces PI3K activation, while the overall expression of the protein remains unaffected within this timeframe . As expeAlthough this work did not directly explore the effect of DMSO on response to treatment(s), data from the “The Genomics of Drug Sensitivity in Cancer” (GDSC) database have provided some insights on their potential interactions. The H2122 cell line, a model whose signaling network was profoundly affected by DMSO, emerged as the most sensitive model across compounds (IC50 Z-score <-2 for 12 of the 180 drugs). In addition, H2122 cells were not resistant to any of the 180 compounds tested. When emphasis was placed on ERK inhibitors exclusively, similar trends were observed. Specifically, cell lines where DMSO induced significant ERK 1/2 downregulation were overall more sensitive to these targeted compounds, while models where ERK 1/2 phosphorylation was unaltered or increased by DMSO showed higher levels of resistance, with only one exception. Although these observations are based on a small number of cell lines, and on a relative short time frame (only up to 24 h), a clear trend was detected between overall drug susceptibility and DMSO effect on cellular signaling networks. This data may have important implications for drug screening and pre-clinical studies, as DMSO may affect drug targets baseline activation levels and susceptibility to treatment. While we specifically designed this analysis to capture short-term adaptation mechanisms associated with DMSO, a few limitations of our work should be addressed. First, this study analyzed in vitro models established from lung malignancies only. Whether these observations hold true across tissue types should be validated in independent analyses. Previous investigations have indicated that the magnitude of the DMSO effects on phospholipids, DNA, RNA and proteins varies from cell type to cell type ,26,27. T2 atmosphere. Cells were passaged using trypsin/EDTA  and sub-cultured at a ratio ranging between 1:3 and 1:10 depending on each cell line’s growth rate.A panel of eight human non-small cell lung cancer cell lines including A549, H1734, H1838, H2122, H21, H358, and H522 were used in this analysis. Calu-3 were purchased from AddexBio , and the remaining seven lines were obtained from American Type Culture Collection . Cell lines were grown following manufacturer’s recommendations. As suggested by the manufacturer, cell lines were grown in media  supplemented with 10% fetal bovine serum (FBS)  at 37 °C and 5% COn = 3) in a 6-well plate and cultured to 80% confluency. Each cell line was treated with three ultra-low DMSO concentrations  0.0008% (C1), 0.002% (C2) and 0.004% (C3) respectively for 5 min, 1 h, 6 h, and 24 h . Baseline untreated control samples were also collected along with the longitudinal samples. At the end of the incubation period, cells were washed twice with PBS  and lysed in TPER  supplemented with 300 mM sodium chloride and a cocktail of protease and phosphatase inhibitors to prevent protein degradation and dephosphorylation including: 1 mM sodium orthovanadate ; 200 mM PEFABLOC ; 5 µg/mL Aprotonin , 5 µg/mL Pepstatin A  (prepared at 1.0 mg/mL in a solution of 10% acetic acid and 90% methanol) and 5 µg/mL Leupeptin  [Cells were seeded in technical replicates  . Proteinn = 3) were averaged so that a single output value was generated for each sample. Coefficient of variations across technical replicates were <5%.Cell lysates were immobilized in technical triplicates onto nitrocellulose coated glass slides  using an Aushon 2470 arrayer  equipped with 185 μm pins. Reference standard curves were printed along with the experimental samples for internal quality control. Selected arrays were stained with Sypro Ruby Protein Blot Stain  following the manufacturer’s directions to quantify the total protein amount for each microarray spot and used for normalization purposes. Immunostaining was performed as previously described . In briehttps://www.cancerrxgene.org, accessed on 1 February 2021). For the work, the GDSC2 dataset released in 2019 was used. As per database instructions, this version of the database drug sensitivity data was collected along with DMSO control data and used for normalization purposes. For each cell line, normalized IC50 are provided for a large panel of targeted compounds and chemotherapeutics. Cell lines were tested with an average of 180 drugs, with the exception of the H1838 and H1734 which were treated with 101 and 29 compounds, respectively. Normalized IC50 scores expressed as Z-scores were retrieved for each cell line. Per database instructions, cell lines with Z-scores > +2 were considered resistant to treatment while those with Z-scores < −2 were classified as sensitive.The effect of DMSO on drug sensitivity was assessed using archived data retrieved from the open-access Genomics of Drug Sensitivity in Cancer (GDSC) database (n = 3) and protein networks were displayed using a graphical presentation where vertices represent individual proteins. Kernel density plots and related histograms were created using R software version 3.4.3. In brief, for each treatment/time pair, replicate measurements were collected . Alpha level for significance was set at 0.05. In order to correct for inflated Type-I error from multiple group comparisons. For each cell line, RPPA values are shown using bar graphs created in GraphPad version v.6.07. RPPA values were first normalized, within each cell line, to baseline level; bar graphs visualize mean and standard error for each group. Venn Diagrams were created in a Gene List Venn Diagram. Pathway enrichment analysis was performed in Reactome Knowledgebase version 75 ,18, and


Canine prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) are typically characterized by metastasis and chemoresistance. Cell lines are important model systems for developing new therapeutic strategies. However, as they adapt to culturing conditions and undergo clonal selection, they can diverge from the tissue from which they were originally derived. Therefore, a comprehensive characterization of cell lines and their original tissues is paramount.This study compared the transcriptomes of nine canine cell lines derived from PAC, PAC metastasis and TCC to their respective original primary tumor or metastasis tissues. Special interests were laid on cell culture-related differences, epithelial to mesenchymal transition (EMT), the prostate and bladder cancer pathways, therapeutic targets in the PI3K-AKT signaling pathway and genes correlated with chemoresistance towards doxorubicin and carboplatin.Independent analyses for PAC, PAC metastasis and TCC revealed 1743, 3941 and 463 genes, respectively, differentially expressed in the cell lines relative to their original tissues (DEGs). While genes associated with tumor microenvironment were mostly downregulated in the cell lines, patient-specific EMT features were conserved. Furthermore, examination of the prostate and bladder cancer pathways revealed extensive concordance between cell lines and tissues. Interestingly, all cell lines preserved downstream PI3K-AKT signaling, but each featured a unique therapeutic target signature. Additionally, resistance towards doxorubicin was associated with G2/M cell cycle transition and cell membrane biosynthesis, while carboplatin resistance correlated with histone, m- and tRNA processing.Comparative whole-transcriptome profiling of cell lines and their original tissues identifies models with conserved therapeutic target expression. Moreover, it is useful for selecting suitable negative controls, i.e., cell lines lacking therapeutic target expression, increasing the transfer efficiency from in vitro to primary neoplasias for new therapeutic protocols. In summary, the dataset presented here constitutes a rich resource for canine prostate and bladder cancer research.The online version contains supplementary material available at 10.1186/s12935-021-02422-9. Prostate cancer in dogs can be classified into two histopathological groups: prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) . Both arMetastasis involves a complex series of events. Among them, the epithelial-to-mesenchymal transition (EMT) plays a critical role in canine prostate cancer , since iChemotherapy can follow tumor type or patient-specific approaches –19. PatiThe present study aims to contribute to the existing knowledge of prostate cancer with the comprehensive transcriptomic characterization of a panel of four PAC, two PAC metastasis and three TCC cell lines and their original tumor tissues. Basic features of this sample set have been reported previously, including medical patient data, histopathology, doubling time, growth behavior, immunophenotype and chemosensitivity towards doxorubicin, carboplatin and meloxicam . ContinuTissue samples and one fine needle-aspiration biopsy of suspected prostate or bladder carcinomas were collected from ten patient dogs at the Small Animal Clinic, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany between 2003 and 2015 with the consent of the patients’ owners  was fresh frozen in liquid nitrogen and stored at −80 °C until RNA isolation. The second part was histopathologically classified into PAC or TCC by a certified and experienced pathologist. Modified Gleason scores were between 8 and 10; metastasis confirmed in nearly all cases . Cell liEach cell line was seeded in T25 flasks in triplicate beyond passage 60. At 70–80% confluency, cells were detached with TrypLE™Express , pelleted, fresh frozen in liquid nitrogen and stored at −80 °C until further use.In accordance with German national legislation, this study does not require ethical approval, as all tissue samples were collected from: (1) a dog that was euthanized due to poor prognosis; or (2) a bladder cancer that was surgically removed as part of treatment; or (3) leftover material from a diagnostic fine needle aspiration biopsy.RNA from cell pellets was isolated using the RNeasy Mini Kit , in accordance with the manufacturer’s protocols. For tissue samples, the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen GmbH) was utilized as previously described . RNA wasSequencing reads were trimmed and filtered using trimmomatic  with paDifferential expression analysis was conducted with the DESeq2 R package . Only prExpression values were obtained by applying the regularized logarithm transformation implemented in the rlog function of the DESeq2 R package  to the rGene expression profiles (z-scores of the regularized-logarithm transformation) were visualized as heatmaps with the pheatmap function from the homonymous R package . Cluster5 cells per insert in serum-free medium. The lower chambers of the wells were filled in duplicates with medium containing 10% serum as attractant, or with serum-free medium as controls. After 48 h of incubation, non-invasive cells were removed from the upper chamber with moistured cotton swabs and two washing steps with PBS. The remaining invasive cells were fixed in 10% formalin for 10 min and permeabilized by methanol for 20 min, both followed by washing twice in PBS. Subsequently, the cells were stained for 2 min with 1% crystal violet, washed three times and allowed to dry. Pictures of three representative areas on each membrane were taken with a DMI600 B microscope  in 100× magnification using the LAS AF 2.6.0 software. Finally, the area covered by invasive cells was quantified using Image J 2.0.0. The experiment was conducted three times independently. Means were compared by univariate ANOVA followed by a Ryan-Einot-Gabriel-Welsch post-hoc test using SAS Enterprise Guide® 7.15.An invasion assay was performed to study EMT and metastatic potential of the nine cell lines. Thereby, cells which were allowed to migrate through an artificial basement membrane, attracted by a serum-containing medium, were quantified. In preparation, cell culture inserts with 8 µm pores in transparent PET membranes  were placed into 12 well plates and coated over night with 200 µg/ml basement membrane extract  in serum-free Medium 199 (Life Technologies GmbH). Additionally, the cells were starved overnight in serum-free medium. The next day, the starved cells were seeded with a density of 2.5 × 10Functional analysis of DEGs was performed with DAVID  , 54, basFor PAC, PAC metastasis and TCC, a gene was considered expressed if it showed a minimum of ten reads in each cell line sample and/or each tissue sample.The fold-changes of the DEGs in the Kyoto Encyclopedia of Genes and Genomes (KEGG) –58 prostAll nine cell lines were washed three times with ice cold PBS and detached by scraping in RIPA buffer containing cOmpleteTM Mini Protease Inhibitor Cocktail . After cell lysis by ultrasound , protein concentrations were determined by Bradford Assay . Next, 30 µg cell lysate per well was separated by electrophoresis in Criterion™ TGX™ gels and blotted onto PVDF membranes using the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories GmbH). The membranes were blocked in Odyssey blocking buffer  diluted 1:3 in PBST for one hour at room temperature. Afterwards, the membranes were incubated with primary antibodies against PI3K-p110α , PI3K-p85 , Akt  and β-Actin   diluted in 1:5 blocking buffer in PBST at 4 °C overnight. The next day, the membranes were washed again, followed by incubation with fluorescence-labeled secondary antibodies  for one hour at room temperature. After three further washing steps, fluorescence signals were detected by a LI-COR Imager with Image Studio Lite software (LI-COR Biosciences GmbH). The Western blot analysis was repeated three times with subsequent passages. Protein lysates of the lymphoma cell lines GL-1 (canine), NALM-6 and SEM (both human) served as positive controls.Human orthologs of canine genes were identified using the table of orthologous genes retrieved from Ensembl BioMart  on NovemHalf maximal inhibitory concentrations of doxorubicin and carboplatin for all nine analyzed cell lines measured in metabolic activity and cell count in a previous study  were corFurthermore, the top 300 genes ranked by ProGENI for doxorubicin or carboplatin resistance based on either metabolic activity or cell count were subjected to functional analysis with DAVID , 54. HumThis study characterized seven cell lines of canine urinary tract cancer in direct comparison to their original tumor tissues. Previous histopathological examination  of the s2 fold-changes, greater than 5 or smaller than −5.Separate analyses for PAC, PAC metastasis, and TCC revealed 1743, 3941 and 463 DEGs, respectively, between the cell lines and their original tumor tissues when controlling for patient-specific effects (Methods). Independently of the histological classification of the samples, biological processes (GO_BP terms) enriched among the identified DEGs were associated with immune response, cell adhesion and angiogenesis/endodermal cell differentiation . Consistent with their high expression of the mesenchymal marker rs Figs.  and 6, Trs Figs. . Also TCrs Figs. B–D. The rs Figs. , were amMMP9 and the group of growth factors and their receptors (GFR), also known as RTK. DEGs in the metastasis cell lines were spread over the entire prostate cancer pathway, including the axes of carcinogenesis, RTKs and downstream molecules, and the hormonal axis  in TCC samples, including the urothelial marker K3A Fig. B. In genThe PI3K-AKT signaling pathway (cfa04151) is an important regulator of the cell cycle and the focus of numerous therapeutic approaches , 21, 68.PI3K and AKT, the key players of the pathway, were not differentially expressed. Moreover, their expression profiles could also be validated at protein level by performing a Western blot . Furthermore, only few downstream genes were differentially expressed, almost exclusively in the PAC metastasis cell lines. Although the functionality of the corresponding proteins warrants further investigation, these findings indicate a good model suitability of the analyzed cell lines for the downstream PI3K signaling cascade. This observation is surprising given that numerous RTKs—direct activators of PI3K—were downregulated. In general, each cell line exhibited its own individual therapeutic marker signature. For instance, the RTK FGFR2 is targeted by erdafitinib, an inhibitor for the treatment of human metastatic bladder cancer [FGFR2 was generally downregulated in PAC cell lines, Adcarc0846 and Adcarc1511.1 appear to have similar FGFR2 expression levels relative to their original tissues. Thus, those two PAC-derived cell lines may still represent suitable model systems for an FGFR2-targeted approach. Furthermore, canine and human TCC tissues and cell lines have been found to overexpress the RTKs EGFR and ERBB2 [EGFR a therapeutic target. A first study has shown promising effects of the EGFR and ERBB2 inhibitor lapatinib on canine TCC cell lines [EGFR inhibitor erlotinib reduced proliferation in a subset of human TCCs [EGFR and ERBB2 were not differentially expressed in the present sample set, suggesting that the characterized cell lines are suitable models for EGFR-targeted therapies. Furthermore, the tyrosine kinases and therapeutical targets EPHA2, MET, LYN and PDGFRA were expressed by specific cell lines at the same level compared to the average of the tissues. Claudins participate in the formation of tight junctions and are therefore essential for the maintenance of an epithelial structure. To promote EMT and metastasis, tight junctions are dismantled in favor of a more invasive, mesenchymal cell type [CLDN1, 3, 4 and 7 and the lowest invasive potential and is hence the most suitable for a claudin-targeted therapy approach. In contrast, Adcarc1258 was highly invasive, expressed all four claudin genes at the lowest level among all cell lines and could therefore be used as negative control.Although cell lines are common model systems for targeted chemotherapy, results obtained with cell lines are not always reproducible in clinical trials . The mair cancer . Althougnd ERBB2 , 89–91. ll lines , while tman TCCs , 94. EGFell type , 95. Theell type , 96. Theell type . Among PCorrelating molecular marker signatures with outcome or chemoresistance can help unravel resistance mechanisms and identify predictive markers  for persLast but not least, this analysis is based on the transcriptome and only selected genes were verified on the protein level. Since translation is influenced by multiple regulatory mechanisms, mRNA levels are not unconditionally transferrable to protein levels . AdditioIn essence, compared to their original tumor tissues, the nine canine cell lines characterized in the present study conserved EMT gene expression programs and downstream PI3K-AKT signaling. Due to their individual therapeutic target signatures, they constitute a suitable panel to evaluate a large variety of inhibitors in vitro. Additionally, the panel includes cell lines derived from primary tumors and metastases of the same patient, which are interesting subjects for metastasis research.Enabled by the rare availability of the tissue samples from which they were derived, this study assessed at the transcriptomic level nine canine PAC, PAC metastasis and TCC cell lines as in vitro models for targeted therapies in canine prostate cancer. Adaptation to culturing conditions and clonal selection resulted in the downregulation of genes associated with tumor microenvironment in the cell lines compared to their original tissues, while tumor- and patient-specific characteristics, as well as the extent of EMT, were mostly conserved. Most importantly, all nine cell lines featured unique therapeutic target expression signatures and preserved subsequent signaling cascades. In summary, this dataset constitutes a rich resource for choosing suitable in vitro models for testing future therapeutic approaches for canine prostate cancer.Additional file 1. Functional analysis of differentially expressed genes (DEGs) and categorization of retrieved enriched biological processes.Additional file 2. The ProGENI top 300 genes associated with doxorubicin and carboplatin resistance quantified by metabolic activity and cell count in the characterized cell lines.Additional file 3. Functional analysis of top 300 genes associated with chemoresistance.Additional file 4. Log2 read counts of therapeutically relevant RTK genes.


Ovarian cancer (OC) is known for its poor prognosis, due to the absence of reliable biomarkers and its late diagnosis, since the early-stage disease is almost asymptomatic. Lipid metabolism plays an important role in OC progression due to the development of omental metastasis in the abdominal cavity. The aim of our study was to assess the therapeutic role of various enzymes involved in lipid metabolism regulation or synthesis, in different subtypes of OC represented by cell lines as well as patient-derived cancer cell cultures (PDCs). We show that proprotein convertase subtilisin/kexin type 9 (PCSK9), a cholesterol-regulating enzyme, plays a pro-survival role in OC and targeting its expression impairs cancer cell growth. We also tested a small library of metabolic and mTOR-targeting drugs to identify drug vulnerabilities specific to various subtypes of OC. Our results show that in OC cell lines and PDCs the second generation of mTOR inhibitors such as AZD8055, vistusertib, dactolisib and sapanisertib, have higher cytotoxic activity compared to the first generation mTOR inhibitors such as rapalogs. These results suggest that, in the era of precision medicine, it is possible to target the metabolic pathway in OC and identify subtype-specific drug vulnerabilities that could be advanced to the clinic.n = 11) and mTOR (n = 10) inhibitors using OC cell lines (n = 8) and ex vivo patient-derived cell cultures  to identify clinically suitable drug vulnerabilities. Targeting PCSK9 expression with siRNA or PCSK9 specific inhibitor (PF-06446846) impaired OC cell survival. In addition, overexpression of PCSK9 induced robust AKT phosphorylation along with increased expression of ERK1/2 and MEK1/2, suggesting a pro-survival role of PCSK9 in OC cells. Moreover, our drug testing revealed marked differences in cytotoxic responses to drugs targeting metabolic pathways of high-grade serous ovarian cancer (HGSOC) and low-grade serous ovarian cancer (LGSOC) PDCs. Our results show that targeting PCSK9 expression could impair OC cell survival, which warrants further investigation to address the dependency of this cancer on lipogenesis and omental metastasis. Moreover, the differences in metabolic gene expression and drug responses of OC PDCs indicate the existence of a metabolic heterogeneity within OC subtypes, which should be further explored for therapeutic improvements. Background: Dysregulated lipid metabolism is emerging as a hallmark in several malignancies, including ovarian cancer (OC). Specifically, metastatic OC is highly dependent on lipid-rich omentum. We aimed to investigate the therapeutic value of targeting lipid metabolism in OC. For this purpose, we studied the role of PCSK9, a cholesterol-regulating enzyme, in OC cell survival and its downstream signaling. We also investigated the cytotoxic efficacy of a small library of metabolic ( Ovarian cancer (OC) continues to be the second most common gynecological cancer in the world, due to its late diagnosis and poor prognosis . MoleculAltered cellular metabolism is one of the hallmarks of tumorigenesis, directly or indirectly connected to the accumulated genomic aberrations . The metA common site for metastatic disease arising from intraperitoneal tumors is the omental tissue which favors metastatic tumor growth . Omentumn = 8) and PDCs (n = 5). Our OC models are comprised of subtype-specific cell lines such as HGSOC and endometrioid, including paired cell-lines pre- and post- acquisition of cisplatin resistance, whereas PDCs were also chosen as representative models of HGSOC and LGSOC. We also analyzed the efficacy of a small library of anti-metabolic (n = 11) and first and second generation of mTOR inhibitors (n = 10) in order to identify metabolic vulnerabilities that could be therapeutically useful in OC treatment.In this study, we investigated the role of PCSK9, the proprotein convertase subtilisin/kexin type-9 regulating cholesterol and lipid metabolism, in OC cell survival and its intracellular signaling, in cell lines . JHOS2 cells were cultured in DMEM/F-12  supplemented with 10% fetal bovine serum, 1× MEM-NEAA  and PrimocinTM . Kuramochi and Ovsaho cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine and PrimocinTM. COV362, HEK293T and HeLa cells were cultured in DMEM (Lonza), supplemented with 10% fetal bovine serum, 2mM L-glutamine and PrimocinTM. All cell lines were kept and incubated at 37 °C and 5% CO2.Human ovarian cancer (OC) cell lines OVCAR3 and OVCAR3cis were cultured in RPMI 1640  supplemented with 20% fetal bovine serum , 2 mM L-glutamine (Thermo Fisher Scientific) and penicillin/streptomycin (Thermo Fisher Scientific). The cisplatin resistant phenotype, OVCAR3cis, cells were cultured with 1 µM cisplatin . The endometrial subtype OC cell lines A2780 and A2780cis, were cultured in RPMI 1640, supplemented with 10% fetal bovine serum, 2 mM L-glutamine and PrimocinSmall interference RNA (siRNA) transfections were performed with Transfection Reagent 1 or 4  cells and with small interference RNA (siRNA) ON-TARGETplus Non-targeting Control Pool (GE Dharmacon) acting as a control or ON-TARGET plus Human PCSK9 siRNA—SMART pool 10 nM (GE Dharmacon) were used to silence PCSK9 in ovarian cancer. The final concentration of siRNA used in transfection was 6 nM for OVCAR3 cells and 25 nM for HeLa cellsTM, Thermo Fisher Scientific). pCINEO expression vector  was used as transfection control.Human PCSK9/NARC1 plasmid (HG15813-CM) was transfected to HEK293T or JHOS2 cells with TurboFect™ Transfection Reagent (Thermo Fisher Scientific) and reduced serum media Opti-MEM I (GibcoCellTiter-Glo (CTG) 2.0 (Promega) assay was used to measure cell viability, according to manufacturer’s instructions. Cells were plated to 96-well plates and untreated or treated with increasing concentrations of the PCSK9 inhibitor (PCSK9i) PF-06446846 hydrochloride  and viability was analyzed after 48 h. The luminescence was measured with Envision plate reader .® 680RD Donkey anti-Goat IgG, IRDye® 800CW Donkey anti-Mouse IgG, or IRDye® 680RD Donkey anti-Rabbit IgG  were used before detection with Odyssey® CLx Imaging System (LI-COR). Image analysis was performed using Image Studio ™ Lite software (LI-COR).Cells were lysed with 50 mM Tris-HCl (pH 7.5) containing 1 mM EDTA, 50 mM NaF, 150 mM NaCl, 1% Triton X-100, 10% glycerol plus 1% phosphatase and protease inhibitor cocktails . Proteins were separated using SDS-PAGE and were transferred onto nitrocellulose membrane . The following primary antibodies were used: anti-PCSK9 antibody , pERK1/2 (T202/Y204) , ERK1/2 , pAKT (Ser473) , AKT , pMEK Ser217/221 , MEK  and anti β-tubulin . Specific LI-COR secondary antibodies IRDyeDrug testing was performed for OC cell lines and PDCs with dose-dependent concentration of drugs for 72 h. Briefly, 1000–1500 cells were added to wells of 384-well plates with drugs pre-plated over a 10,000-fold concentration range (in five concentrations). After a 3-day incubation at 37 °C, cell viability was measured using the Cell Titer-Glo reagent (Promega) and plates were read with PHERAstar FS . For each drug, dose–response curves were generated and DSS were calculated as previously described . In shorTotal RNA was isolated from PDCs using total RNeasy kit . Quantity and quality of the RNA samples were assessed by Qubit  and Bioanalyzer . RNA with an RNA integrity number (RIN) > 8 was used to derive libraries and the sequencing was performed with Illumina HiSeq system  as previously described . The expPCSK9 increases LDLR degradation and, therefore, decreases the circulating LDL uptake and clearance [Proprotein convertases (PCs) are serine proteases capable of specific proteolysis at the RXR/KR motif in a multitude of substrates such as growth factors and growth factor receptors and pro-angiogenic proteins . This falearance . Since lTo date, there are not any known LGSOC representative cell lines. Moreover, for OVCAR3 and A2780 cell lines, we have included both parental and cisplatin-resistant clones, as platinum chemoresistance usually develops in OC patients and has therapeutic significance. Western blot analysis of OC cell line lysates identified variable expression of PCSK9 isoforms (uncleaved and cleaved) in comparison with cervical carcinoma HeLa cell line, which was previously shown to express PCSK9  Figure . The higAPOA1, APOA4, APOH, APOC3 as well as PCSK2, PCSK6 and PCSK1, suggesting a subtype specific metabolic gene expression profile of OC PDCs.Furthermore, we wanted to evaluate PCSK9 expression in OC tumor samples and for this, we used PDCs derived from HGSOC and LGSOC patient tumors. Personalized cancer therapy using ex vivo pharmacogenomic testing of patient-derived cancer cells has already shown great potential and PDCs are currently used as models for tumor molecular profiling as excellent ex vivo representatives of patients’ tumor . We asseNext, we wanted to assess whether targeting PCSK9 expression has any effect on OVCAR3 and HeLa cell viability. siRNA-mediated knockdown of PCSK9 expression resulted in a statistically significant reduction of cell survival A, and a We also investigated whether overexpression of PCSK9 in JHOS2 cells that typically lack its expression will activate pro-survival signaling pathways such as AKT/ERK. Transient transfection of PCSK9 in JHOS2 cells resulted in increased phospho-AKT, phospho-ERK and phospho-MEK levels, as well as an increase in ERK and MEK basal expression . This co2), an alcohol dehydrogenase inhibitor chelating copper ions selectively accumulated in cancer cells. We also focused on compounds that inhibit DNA or RNA synthesis by blocking the activity of various enzymes required for metabolic reactions, such as daporinad, AVN944, methotrexate, pemetrexed, TH588 and triapine  pathway helped us to understand the molecular mechanisms that regulate eukaryotic cell growth, linking nutrient sensing to regulation of protein synthesis, metabolism, aging and disease development . mTOR isTargeting the mTOR pathway has been extensively explored in cancer therapy and the first mTOR inhibitors approved were a class of rapamycin derivatives known as “rapalogs”. Rapamycin, also known as sirolimus and one of the first generation mTOR inhibitors, exerts its effect by binding to FRBP-12 (12 kDa FK506-binding protein) to form a ternary complex with mTOR, leading to inactivation of mTORC1 . Other rMany human diseases, including cancer, are the consequence of altered metabolic pathways. Cancer cells have distinctive metabolic phenotypes characterized by altered nutrient metabolism that supports the rapid manufacture of biomass required to sustain rapid cell proliferation . OC is pIn this study, we investigated expression levels and an anti-apoptotic role of PCSK9 in OC models including cell lines and PDCs from various subtypes of OC. We found variable PCSK9 expression in our OC cell lines, with OVCAR3 showing the highest level of PCSK9  compared to HeLa cells that are known to express PCSK9 A,B. DeteAPOA1, APOA4, APOH, APOC3 as well as PCSK2, PCSK6, PCSK1 were observed in LGSOC PDCs compared to HGSOC PDCs  is an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT) specifically inhibiting the biosynthesis of NAD+ from niacinamide, which is essential for the cellular metabolism, protein modification and messenger synthesis . DaporinAmong the mTOR targeted drugs, our screen showed that the first generation of rapalogs had overall low DSS values in both OC cell lines and PDCs, except for PDC#4 which displayed good sensitivity to all rapalogs B. PrevioOur study is the first to evaluate the expression and anti-apoptotic role of PCSK9 in OC. By investigating the efficacy of various metabolic and mTOR inhibitors ex vivo using tumor representative OC PDCs, our results showed differences in drug cytotoxic responses in LGSOC compared to HGSOC PDCs, which is in line with previous studies showing that OC subtypes display clear metabolic phenotypes . As such