The xCELLigence system revealed that scoulerine exerted potent antiproliferative activity in lung, ovarian and breast carcinoma cell lines. Jurkat and MOLT-4 leukemic cells treated with scoulerine were decreased in proliferation and viability. Scoulerine acted to inhibit proliferation through inducing G2 or M-phase cell cycle arrest, which correlates well with the observed breakdown of the microtubule network, increased Chk1 Ser345, Chk2 Thr68 and mitotic H3 Ser10 phosphorylation. Scoulerine was able to activate apoptosis, as determined by p53 upregulation, increase caspase activity, Annexin V and TUNEL labeling. Results highlight the potent antiproliferative and proapoptotic function of scoulerine in cancer cells caused by its ability to interfere with the microtubule elements of the cytoskeleton, checkpoint kinase signaling and p53 proteins. This is the first study of the mechanism of scoulerine at cellular and molecular level. Scoulerine is a potent antimitotic compound and that it merits further investigation as an anticancer drug.Scoulerine is an isoquinoline alkaloid, which indicated promising suppression of cancer cells growth. However, the mode of action (MOA) remained unclear. Cytotoxic and antiproliferative properties were determined in this study. Scoulerine reduces the mitochondrial dehydrogenases activity of the evaluated leukemic cells with IC Natural isoquinoline alkaloids as contained in plant extract remedies have been used in traditional medicine for centuries  and have wide-ranging properties that play an important role in the human combat against diseases. Strangely, although various plant families have been extensively investigated in search for constituents with a therapeutic significance, the alkaloids found in the Papaveraceae family plants have not been well studied so far. Among the Papaveraceae alkaloids which are known to possess some bioactive properties, scoulerine (1)  and K1CB1 (multidrug resistant strain), with IC50 values 1.78 µg/mL and 1.04 µg/mL, respectively. Regrettably, this activity does not meet the criteria stipulated under the Medicines for Malaria Venture3. Other research efforts, performed on rats, determined that scoulerine protects α-adrenoreceptors against irreversible blockade by phenoxybenzamine, inhibits [3H]-inositol monophosphate formation caused by noradrenaline8 and acts as a selective α1D-adrenoreceptor antagonist without affecting the contraction of the rat aorta9. Scoulerine has also been reported to exhibit other useful pharmacological properties such as antiemetic, antitussive and antibacterial action3 and has been found to have an affinity to the GABA receptors2. Interestingly, a pioneer cell culture study on this alkaloid described that scoulerine shows significant cytotoxic activity against A549 and HT-29 cancer cell lines. The authors imply that the cytotoxic potency of scoulerine is associated with its ability to stabilize the covalent topoisomerase I - DNA complex to promote the formation of single-strand DNA breaks10. It should be pointed out that the unique position of scoulerine in backbone arrangements during biosynthesis and its interesting biological activities already attracted our attention in two previous studies. Scoulerine was found to be active as an inhibitor of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), which is a very promising target for the treatment of Alzheimer’s disease (AD)5. In our follow-up work, when considering forty-six isoquinoline alkaloids screened by MTT assay, scoulerine exhibited impressive cytostatic activity against gastrointestinal cancer cells11. Although our recent study demonstrated the bioactivity of scoulerine with an emphasis on the cytostatic action that may be of interest in cancer chemotherapy, further studies remain to be undertaken to better explore its anticancer potential. At present, this study provides a better investigation of the MOA of scoulerine at cellular and molecular level. In addition to that, the pro-apoptotic and cell cycle arrest activity in p53-deficient (Jurkat) and p53 wild-type (MOLT-4) leukemic cells following scoulerine treatment is determined. Finally, aiming at the further conceptual extension to study structure-cytotoxicity relationships, we have introduced three  aliphatic derivates of scoulerine incorporating esters of carboxylic acids.Plant natural compounds and their derivatives continue to provide an indispensable source of new drug leads for drug development. In the area of cancer therapy, up to 80% of approved drugs are either natural products per se or are based thereon1) and its three semi-synthetic derivatives (2), (3) and (4) were evaluated at a single-dose exposure (10 µM) against a mini-panel of leukemic cell lines  using the XTT metabolic activity assay ,  and (4) In what concerns the pro-apoptotic activity of scoulerine in both Jurkat and MOLT-4 leukemic cell lines, this compound showed an apoptosis-inducing effect assayed by Annexin V and PI staining 24 h following the treatment. Soon after initiating apoptosis, the membrane phosphatidylserine was translocated from the inner layer of the plasma membrane to the outer leaflet. Quantification of Annexin V binding to externalized phosphatidylserine allowed us to clearly identify apoptotic cells between cell populations. Twenty-four hours after the application of 0, 2.5, 5, 10, 15 and 20 µM of scoulerine, the early apoptotic rates were 4%, 8%, 22%, 24%, 22% and 23% for Jurkat cells, and 3%, 4%, 9%, 16%, 14% and 13% for MOLT-4 cells, respectively. The late apoptotic  rates for Jurkat leukemic cells were 5%, 15%, 21%, 21%, 19% and 22%, respectively, and 5%, 14%, 20%, 27%, 26% and 28% for MOLT-4, respectively  activation of caspases-3/7 and -8, and a bit less pronounced (P ≤ 0.05) activation of caspase-9. At the 48 h interval, the activity of caspases-3/7, -8 and -9 was still statistically significant, but began to decline. On the contrary, where MOLT-4 leukemic cells were concerned, the activity of caspases-3/7, -8 and -9 was even more pronounced 48 h following the application of 2.5 and 5 µM of scoulerine, compared to the same after 24 h, with an overall higher level of activity of caspases-3/7, -8 and -9  to 29% and 22%, respectively. Conversely, the proportion of cells in the G2/M phase increased from 24% (mock-treated control) to 49%. The rise in the percentage of Jurkat cells in the G2/M phase was even more eminent in the presence of 10, 15 and 20 µM of scoulerine. A similar trend was observed in MOLT-4 cells treated with the same amounts of scoulerine  measured as positive for H3 pSer10 staining using flow cytometry revealed that scoulerine-treated Jurkat (2.5–20 µM) and MOLT-4 (5–20 µM) cells for 24 h were significantly (P ≤ 0.05) arrested in mitosis. Together, these results indicate that scoulerine is able to induce cell cycle arrest in the G2 or M phase depending on the dose and the duration of the treatment Fig. .Figure 7Since we observed significant G2 or M phase arrest and H3 pSer10 staining during flow cytometry cell cycle analysis, we explored whether scoulerine affects the microtubule structures of cells. To visualize this, lung carcinoma A549 cells were chosen as a model for indirect immunofluorescence with a monoclonal anti-β-tubulin antibody due to a lower nuclear-cytoplasmic ratio compared to leukemic cells. Additionally, A549 cells were sensitive to scoulerine and displayed strongly diminished proliferation during xCELLigence analysis at concentrations exceeding 10 µM. Epi-fluorescence microscopy performed with fixed A549 cells labeled with a monoclonal anti-β-tubulin antibody and DAPI counter stain revealed that the intact microtubules extended continuously through the cytoplasm in control cells. In contrast, scoulerine applied for 24 h at 10 and 20 µM obviously disrupted microtubule structure with a dense aggregation of microtubules around cell nuclei, being less dense at the cell periphery. Besides, A549 cells treated with scoulerine at 5 µM resisted the treatment with almost intact organized tubulin networks as seen in 0.1% DMSO exposed control cells. Nocodazole, the microtubule depolymerizer, was used as a positive control for the study. As is apparent from the microscopic images presented in Fig. To assess DNA damage, the alkaline comet assay was performed. The treatment of Jurkat and MOLT-4 cells with scoulerine at 2.5 and 5 µM for 24 h, triggered apoptotic (secondary) DNA fragmentation, which was estimated by the formation of comet-like tails after single-cell gel electrophoresis. Briefly, in a 24 h interval, there was a significant increase in the tail moment after scoulerine application in both Jurkat and MOLT-4 leukemic cells. However, when we performed the comet assay at an earlier 12 h interval of treatment, no considerable direct DNA damage was found. This suggests that DNA damage is predominantly a consequence of internucleosomal cleavage associated with apoptotic cell death in response to scoulerine treatment Fig. .Figure 9In order to study the molecular mechanism by which scoulerine induces cell cycle arrest and apoptosis, we investigated the expression levels and the activation of several cell cycle regulatory and proapoptotic proteins. To perform this, Jurkat and MOLT-4 cells were treated with scoulerine at 2.5 and 5 µM for 24 h and whole cell lysates were prepared and used for Western blotting. Western blot analysis showed an upregulation of p53 protein in p53 wild-type MOLT-4 cells in the presence of 5 µM scoulerine. In Jurkat cells, checkpoint kinase 1 (Chk1) was activated through phosphorylation at Ser345 and checkpoint kinase 2 (Chk2) was activated through phosphorylation at Thr68 after scoulerine treatment. However, in both determined cell lines the expression levels of p21, which is also involved in the regulation of cell cycle progression, remained the same. Similarly, the level of phosphorylated p53 at Ser15, which increased after cisplatin exposure (positive control), remained unchanged after scoulerine exposure Fig. .Figure 111. Another recent work in this direction showed that scoulerine isolated from the stems of Xylopia laevigata was cytotoxic toward the tumor cell lines B16-F10, HepG2, K562 and HL-6012. Encouraging results prompted us to investigate whether scoulerine can eliminate cancer cells via apoptosis and if the scoulerine-induced antiproliferative effect blocks cell cycle progression. Thus, in the work herein, we have investigated proliferation, cell cycle distribution, cell death, apoptosis induction, DNA damage, microtubule structure and the upregulation of selected DNA-damage response proteins following scoulerine treatment. We show that scoulerine had cytostatic activity in all of the leukemic and tumor lines investigated in a dose-dependent manner. Controversially, our results are in contrast with that reported by Khamis and colleagues. They determined only moderate cytotoxic activity of discretamine (scoulerine) with IC50 over 3000 µM using four human breast cancer  cell lines and MTT assay13. Here, however, scoulerine inhibited the proliferation of MCF-7 cells at 10 µM, as measured by means of the xCELLigence system in view of the cell-growth inhibition profile under real-time. To better understand antiproliferative potential of this naturally occurring alkaloid, derivatives of scoulerine were synthesized and assayed at 10 µM with respect to its activity on cell growth. The comparison of the semisynthetic derivatives (2), (3) and (4) with scoulerine indicated that esterification generally resulted in a reduction in the antiproliferative activity. Moreover, it seems that the potencies of the (2), (3) and (4) esters decreased with an increasing length of the carbon chain. Next, we observed that scoulerine treatment significantly downregulated the viability of Jurkat and MOLT-4 cells within 48 h from 5 μM and higher as assessed by using a Trypan blue exclusion test. Apoptosis induction associated with the translocation of phosphatidylserine to the outside of the cell and loss of plasma membrane integrity was quantified by flow cytometry. Jurkat and MOLT-4 cell cultures treated with scoulerine showed a presence of both early and late apoptotic cells, which increased in a dose-dependent manner. Apoptosis induction was further supported by measuring DNA fragmentation using TUNEL assay, by quantifying caspase activity and by detecting p53 expression in MOLT-4 cells. Collectively, scoulerine showed caspases-3/7, -8 and -9 activation, which suggests the involvement of both an extrinsic and intrinsic pathway of programmed cell death. Further analysis by flow cytometry revealed that scoulerine treatment led to G2 phase arrest in Jurkat and MOLT-4 leukemic cells followed by an increase in the percentage of cell population in mitosis as judged by PI and phospho-histone H3 double staining. During mitotic division, the tubulin-microtubule protein system underwent assembly, which, collectively, is an essential component for chromosome segregation. Where antimitotic compounds are concerned, this protein system is a suitable target. It is these antimitotic compounds that interfere with the microtubules function by inhibiting cell proliferation in mitosis14. Since uncontrolled and rapid cell division is a hallmark of cancer tumor growth, microtubule-interfering agents display a remarkable efficacy in rapidly proliferating cancer cells15. Therefore, we sought to elucidate, using immunofluorescence imaging, whether treatment with scoulerine affects the cytoskeletal microtubule architecture of cells. The epi-fluorescence images obtained, where A549 cells were exposed to 10 and 20 μM of scoulerine for 24 h and stained with an anti-β-tubulin antibody, revealed a disruption of the tubulin structure. These results support the notion that scoulerine is a mitotic poison, resulting in G2 or M arrest. Unlike drugs grouped as genotoxic anticancer agents, our results indicate that scoulerine does not interfere with DNA, thereby activating cancer cell death independently of exogenous-induced DNA damage. However, these findings oppose what has been reported by Cheng, who proposed that scoulerine’s anticancer activity is associated with the formation of single-strand DNA breaks through the stabilization of the covalent topoisomerase I – DNA complex10. Thus, dissimilarly to the previous findings describing the DNA-damaging action of scoulerine, no significant DNA damage was found early at 12 h using the alkaline comet assay. However, as we expected, the later 24 h interval of treatment induced DNA fragmentation that resulted from an internucleosomal cleavage of DNA during scoulerine-induced apoptosis. Such single- or double-strand DNA breaks generated during apoptotic cell death were cross-verified by TUNEL at 24 h in both leukemic cell lines. Numerous reports have shown that replication stress and DNA damage activate critical cell-cycle checkpoint kinases Chk1 and Chk2 of the DNA damage response pathway17. Both Chk1 phosphorylated at Ser345 by Ataxia telangiectasia and Rad3-related (ATR) kinase and Chk2 phosphorylated at Thr68 by Ataxia telangiectasia mutated (ATM) kinase were determined after 24 h of treatment. These results imply that activated Chk1 and Chk2 are particularly important to pause cell cycle progression after scoulerine exposure.In our previous study we reported that scoulerine has a potent antiproliferative activity against Caco-2 and Hep-G2 cancer cellsS)-norcoclaurine as the central precursor for the biosynthesis. Internal carbon-carbon coupling of the (S)-norcoclaurine derivative results in the formation of branch point intermediate (S)-reticuline. Then stereo-specific conversion changes of the central intermediate (S)-reticuline to (S)-scoulerine and the formation of (S)-scoulerine is the first step in the biosynthesis of the subsequent benzo[c]phenanthridine, protopine and protoberberine alkaloids, sanguinarine, protopine and berberine, respectively18. Among these, it is important to point out that sanguinarine, protopine and berberine were reported to show various degrees of cytotoxicity and antiproliferative activity against cancer cells. Although sanguinarine did not change the proportion of mitotic cells, it inhibited tubulin polymerization leading to the disruption of the microtubule network19. Interestingly, protopine is able to target microtubule structures in living cells without affecting tubulin polymerization with cell cycle arrest at G2/M20. In the same manner, disruptions of the microtubule network were described in response to berberine treatment21. Even if this structurally related substance shares some mechanistic similarities, it should be noted that these compounds differ in growth inhibitory values and molecular actions, thus making them an attractive target for structure-activity relationship studies.Although scoulerine has been studied for several years, only few publications regarding its cytotoxic activity against mammalian cells exist. The most explored part is its biosynthesis in the plants of the Fumaridaceae family. The biosynthesis of scoulerine begins with the condensation of two tyrosine derivatives, dopamine and 4-hydroxyphenylacetaldehyde, yielding 1-benzylisoquinoline  spectrometer operating at 500 MHz for 1H and 125 MHz for 13C. Chemical shifts were recorded as δ values in parts per million (ppm), and were indirectly referenced to tetramethylsilane (TMS) via the solvent signal . Coupling constants (J) are given in Hz. For unambiguous assignment of 1H and 13C signals 2D NMR spectra  were measured using standard parameter settings and pulse programs delivered by the producer of the spectrometer. ESI-HRMS were obtained with a Waters Synapt G7-Si with a hybrid mass analyzer quadrupole-time-of-flight (Q-TOF), coupled to a Waters Acquity I-Class UHPLC system. The EI-MS were obtained on an Agilent 7890 A GC 5975 inert MSD operating in EI mode at 70 eV . A DB-5 column  was used. The temperature program was: 100–180 °C at 15 °C/min, 1 min hold at 180 °C, and 180–300 °C at 5 °C/min and 5 min hold at 300 °C; detection range m/z 40–600. The injector temperature was 280 °C. The flow-rate of carrier gas (helium) was 0.8 mL/min. A split ratio of 1:15 was used. TLC was carried out on Merck precoated silica gel 60 F254 plates. Compounds on the plate were observed under UV light (254 and 366 nm) and visualized by spraying with Dragendorff´s reagent.All reagents were purchased from commercial sources  and used without purifications. NMR spectra were recorded for CDCl1) has been previously isolated from dry tubers of Corydalis cava4. Results of GC/MS, HPLC, 1H-NMR and 13C-NMR analysis are shown in Supplementary Figs Scoulerine (1) in 3 mL of dry pyridine. The mixture was stirred at room temperature until disappearance of the starting material. Then, the solvent was evaporated and the residue was purified by preparative TLC using cHx:Et2NH 9:1 or cHx:To:Et2NH 45:45:10 to afford corresponding esters 2–422.The 1.5 equiv. of the corresponding anhydride was added to a solution of scoulerine  were obtained as an orange oil. 1H NMR  δ: 7.01 , 6.93 , 6.84 , 6.71 , 4.03 , 3.83 , 3.82 , 3.59 , 3.45 , 3.23 , 3.21–3.12 , 2.86 , 2.79–2.70 , 2.69–2.61 , 2.35 , 2.33 . 13C NMR  δ: 169.2, 168.6, 149.2, 149.0, 138.0, 136.1, 133.0, 130.0, 128.1, 127.5, 126.4, 119.7, 112.3, 110.6, 58.6, 56.0, 55.9, 53.3, 51.0, 35.8, 29.4, 20.7, 20.4. [α]24D = −198 ; EIMS m/z (%) 411 (41), 410 (52), 352 (50), 176 (23), 150 (100). HRESIMS m/z calcd for C23H26NO6 (M + H+) 412.1760 found 412.176. Results of GC/MS, HPLC, 1H-NMR, 13C-NMR and HRMS analysis are shown in Supplementary Figs Following the procedure described above, 17 mg (0.05 mM) of 1 was treated with 0,2 mL of propionic anhydride. After purification, 21 mg of 3 (60%) were obtained as an orange oil. 1H NMR  δ: 7.00 , 6.92 , 6.83 , 6.70 , 4.01 , 3.81 , 3.80 , 3.58 , 3.44 , 3.23 , 3.21–3.11 , 2.85 , 2.76–2.69 , 2.68–2.61 , 2.65 , 2.63 , 1.32 , 1.30 . 13C NMR  δ: 172.7, 172.1, 149.2, 149.0, 138.1, 136.1, 132.9, 130.0, 128.1, 127.5, 126.3, 119.7, 112.2, 110.5, 58.6, 56.0, 55.9, 53.3, 51.1, 35.8, 29.4, 27.3, 27.2, 9.3, 9.1. [α]24D = −147 ; EIMS m/z (%) 439 (21), 438 (46), 382 (14), 366 (66), 326 (9), 176 (25), 149 (100). HRESIMS m/z calcd for C25H30NO6 (M + H+) 440.2073 found 440.2071. Results of GC/MS, HPLC, 1H-NMR, 13C-NMR and HRMS analysis are shown in Supplementary Figs Following the procedure described above, 26 mg (0.08 mM) of 1 was treated with 0,2 mL of hexanoic anhydride. After purification, 23 mg of 4 (76%) were obtained as an orange oil. 1H NMR  δ: 7.00 , 6.91 , 6.83 , 6.70 , 4.01 , 3.81 , 3.80 , 3.59 , 3.44 , 3.23 , 3.21–3.11 , 2.86 , 2.77–2.69 , 2.68–2.64  2.61 , 2.59 , 1.85–1.76 , 1.49–1.36 , 0.95 . 13C NMR  δ: 172.1, 171.4, 149.2, 149.0, 138.1, 136.1, 132.8, 130.0, 128.1, 127.4, 126.3, 119.7, 112.2, 110.5, 58.6, 56.0, 55.8, 53.4, 51.1, 35.8, 34.0, 33.9, 31.3, 31.2, 29.4, 24.8, 24.7, 22.3, 13.9, 13.9. [α]24D = −200 ; ESIMS m/z (%) [M + H+] 524 (100). HRESIMS m/z calcd for C31H42NO6 (M + H+) 524.3012 found 524.3013. Results of GC/MS, HPLC, 1H-NMR, 13C-NMR and HRMS analysis are shown in Supplementary Figs Following the procedure described above, 19 mg  of The purity of all compounds verified by NMR was ≥97%.2–95% air. Cells were passaged every 2–3 days to obtain exponential growth. The cells were maintained in culture no more than 20 passage.Jurkat, Raji and A2780 cells were propagated in RPMI 1640 medium supplemented with 10% foetal bovine serum, 2 mM L-glutamine, 1 mM pyruvate, 10 mM HEPES, MEM Non-Essential Amino Acids 10 µl/ml, 50 µg/ml penicillin and 50 µg/ml streptomycin . MOLT-4 cells were cultured in RPMI 1640 medium supplemented with 20% foetal calf serum, 2 mM L-glutamine, 1 mM pyruvate, 10 mM HEPES, MEM Non-Essential Amino Acids 10 µL/mL, 50 µg/mL penicillin and 50 µg/mL streptomycin . HEL 92.1.7 cells were cultured in RPMI 1640 medium supplemented with 10% foetal calf serum, 2 mM L-glutamine, 50 µg/mL penicillin and 50 µg/mL streptomycin . HL-60 cells were cultured in RPMI 1640 medium supplemented with 20% foetal calf serum, 2 mM L-glutamine, 50 µg/ml penicillin and 50 µg/ml streptomycin . U-937 cells were cultured in RPMI 1640 medium supplemented with 10% foetal calf serum, 2 mM L-glutamine, 1 mM pyruvate, 10 mM HEPES, 50 µg/ml penicillin and 50 µg/ml streptomycin . A549 cells were cultured in Minimum Essential Medium Eagle with L-glutamine and sodium bicarbonate  in the presence of 10% foetal calf serum, 1 mM pyruvate, 10 mM HEPES, 50 µg/ml penicillin and 50 µg/ml streptomycin . SK-BR-3 cells were cultured in McCoy’s 5 A medium  supplemented with 10% foetal bovine serum and 50 µg/ml penicillin/streptomycin . MCF-7 cells were maintained in Minimum Essential Medium Eagle with L-glutamine and sodium bicarbonate  supplemented with 10% foetal calf serum, 1 µg/ml insulin, 50 µg/ml penicillin and 50 µg/ml streptomycin . Jurkat, MOLT-4, Raji, HL-60, U-937, HEL 92.1.7, A549, A2780, SK-BR-3 and MCF-7 cells were purchased from the European Collection of Cell Cultures . The cell cultures were maintained under standard cell culture conditions at 37 °C in a humidified incubator in an atmosphere of 5% COCorydalis cava (L.) Schweigg. et Koerte (Fumariaceae). The detailed isolation and identification  of alkaloid is described in4. Esters of (-)-scoulerine were dissolved in dimethyl sulfoxide - DMSO  at 50 mM. For the experiments, the stock solutions were diluted with the complete culture medium to create final concentrations of 1–50 μM, making sure the concentration of DMSO was <0.1% to avoid the toxic effects on the cells. Cisplatin, doxorubicin and nocodazole were purchased from Sigma-Aldrich . Control cells were sham-treated with a DMSO vehicle only . Cells treated with 5% DMSO, cisplatin at 5 µM, doxorubicin at 1 µM or nocodazole at 5 µM were used as positive control.Scoulerine; (−)-scoulerine: fresh stock solutions of scoulerine in concentrations of 50 mM were dissolved in dimethyl sulfoxide - DMSO . Stock solutions were freshly prepared before use in the experiments. Scoulerine was isolated from tubers of 23. Data were analysed with GraphPad Prism 5 biostatistics  statistical software. Each value is the mean of at least three independent replicates of each condition.In order to determine cell viability of cells treated with scoulerine, alkaloid esters (a single-dose of 10 µM) or scoulerine in a broad concentration range we used a standard colorimetric method measuring a tetrazolium salt reduction via mitochondrial dehydrogenase activity. The cells were seeded at previously established optimal density in a 96-well plate. After 48 hours incubation cell viability was determined using Cell Proliferation Kit II  according to manufacturer’s instructions. XTT-assay was conducted using 200 μl of volume and 100 μl of XTT-labeling mixture. Absorbance was then measured at 480 nm using a 96-multiwell microplate reader Tecan Infinite M200 . Viability was calculated as described in the paper by Havelek and colleagues using the following formula: (%) viability = (A480sample − A480blank)/(A480control − A480blank) × 100, where A480 is the absorbance of utilized XTT formazan measured at 480 nm2. First, the optimal seeding concentration for experiments was optimized for each cell line. After seeding, the respective number of cells in 190 µL medium per each well of the E-plate 96, the proliferation, attachment and spreading of the cells were monitored every 30 minutes by the xCELLigence system. Approximately 24 h after seeding, when the cells were in the log growth phase, the cells were exposed in triplicates to 10 µL sterile deionized water containing scoulerine to obtain final concentrations 1–50 μM. Controls received sterile deionized water + DMSO with a final concentration of 0.1%. Cells treated with 5% DMSO were used as positive control. Growth curves were normalized to the time point of treatment. Evaluations were performed using xCELLigence 1.2.1 software .The xCELLigence system  was used to monitor cell adhesion, proliferation and cytotoxicity. The xCELLigence system was connected and tested by Resistor Plate before the RTCA Single Plate station was placed inside the incubator at 37 °C and 5% COCell proliferation and viability of Jurkat and MOLT-4 cells were determined 24 and 48 h after treatment with 2.5, 5, 10, 15 and 20 μM of scoulerine. Cells treated with 5 µM cisplatin were used as positive control. Cell membrane integrity was determined using the Trypan blue exclusion technique – mixing 10 μl of 0.4% Trypan blue and 10 μl of cell suspension. Cell counts were carried out using a Bürker chamber and light microscope Nikon Eclipse E200 .2+, and the property of propidium iodide (PI) to enter cells with damaged cell membranes and to bind to DNA24. Measurement was performed immediately using a CyAn  flow cytometer. Listmode data were analysed using Kaluza Analysis 1.3 software .Apoptosis was determined by flow cytometry using an Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis kit  according to the manufacturer’s instructions. The Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis kit employs the property of Alexa Fluor® 488 conjugated to Annexin V to bind to phosphatidylserine in the presence of Ca6 cells were collected and evaluated using a DNA breaks labelling detection kit, APO-DIRECT  in accordance with the manufacturer’s instructions25. Cells were washed twice with PBS at 4 °C. Then, 1 mL of fixation solution was added to the cell suspension  for 30 min on ice. Cells were centrifuged  and washed twice with 5 mL PBS. Cells were permeabilized in 70% ethanol for at least 24 h at −20 °C. Cells were washed twice with wash buffer and incubated in a TUNEL reaction mixture  for 60 min at 37 °C. Cells were washed again twice with rinse buffer and resuspended in 0.5 mL propidium iodide/RNase A solution. Samples were analysed in a CyAn ADP  flow cytometer, measuring green (dUTP-FITC incorporated in fragmented DNA) and red (PI binding to DNA) fluorescence of nuclei of individual cells. Listmode data were analysed using Kaluza Analysis 1.3 software .To determine apoptosis by TUNEL assay, 1 × 102HPO4 + 8 ml of 0.1 M citric acid, pH 7.8) and then labelled with propidium iodide in Vindelov’s solution for 1 h at 37 °C24. DNA content was determined using the flow cytometer CyAn  with an excitation wave length of 488 nm. The data were analysed using Multicycle AV software .Where cell cycle distribution analysis is concerned, the cells were washed with ice cold PBS and fixed with 70% ethanol. In order to detect low molecular-weight fragments of DNA, the cells were incubated for 5 minutes at room temperature in a buffer . After treatment, the Caspase-Glo Assay Reagent was added to each well (50 μl/well) and incubated for 30 minutes before luminescence was measured using a Tecan Infinite M200 spectrometer .The induction of programmed cell death was determined by monitoring the activities of caspases 3/7, caspase 8 and caspase 9 by Caspase-Glo Assays  24 and 48 h after treatment with 2.5 and 5 μM of scoulerine. Cells treated with 5 µM cisplatin were used as positive control. The assay provides a proluminogenic substrate in an optimized buffer system. The addition of a Caspase-Glo Reagent results in cell lysis, followed by caspase cleavage of the substrate and the generation of a luminescent signal. A total of 1 × 10Histone H3 is phosphorylated at Ser10 when cells enter the mitotic phase and remains unphosphorylated in the other phases of the cell cycle. To determine the number of mitotic cells, cells treated with scoulerine for 24 h were fixed and permeabilized by adding ice-cold 100% methanol. Following overnight incubation, the cells were washed with a PBS-0.5% BSA solution and subsequently stained with the Alexa Fluor 488-conjugated anti-pSer10-histone H3  antibody diluted with a PBS-0.5% BSA solution in accordance with the manufacturer’s instructions. Cells were incubated with agitation for 60 minutes at room temperature in the dark. Finally, the cells were washed twice with the PBS-0.5% BSA solution and resuspended in a PI staining solution  at a final volume of 500 µL. All samples were immediately analyzed by a CyAn flow cytometer and the data were plotted using Kaluza Analysis 1.3 software .For each condition, 250 000 cells were seeded in 2-well chamber slides SPL . After seeding , spent medium was replaced with fresh medium and the cells were treated with scoulerine at 5, 10 and 20 µM. Cells treated with 5 µM nocodazole were used as positive control. Following 24-h treatment the cells were fixed with 4% freshly prepared paraformaldehyde for 10 minutes at room temperature, washed with PBS, permeabilized in 0.2% Triton X-100/PBS for 15 minutes at room temperature and washed with PBS . Before incubation with the primary antibody (overnight at 4 °C), the cells were incubated with 7% heat inactivated foetal calf serum +2% bovine serum albumin in PBS for 30 minutes at room temperature. Mouse monoclonal anti-β-tubulin  was used for β-tubulin detection. For the secondary antibody, the affinity pure donkey anti-mouse-TRITC-conjugated antibody was purchased from the Jackson ImmunoResearch Laboratories . The secondary antibody was applied to each slide (after their pre-incubation with 5.5% donkey serum in PBS for 30 minutes at room temperature), incubated for 1 h in the dark and washed (3 × 5 min) with PBS. The nuclei were counterstained with 100 µl of 4′,6-diamidino-2-phenylindole (DAPI) at 1 µg/ml for 30 min. After the last two washes with PBS, the slides were mounted with an antifading ProLong® Gold mounting medium . Images of all of the examined slides were obtained by a Nikon epi-fluorescence microscope system Eclipse 80i; the exposure time and dynamic range of the camera in all of the channels were adjusted to the same values for all of the slides to portray quantitatively comparable images. Images were further processed and merged using NIS-Elements Advanced Research 4.13 .DNA damage was measured using alkaline version of comet assay. Briefly, cells embedded in 1% agarose  on microscope slides were lysed in 10 mM Tris-buffered 2.5 M NaCl  containing 1% Triton × 100  and 100 mM EDTA  for 1 hour at 4 °C. In alkaline conditions , the electrophoresis was carried out at 40 V, 300 mA, for 30 minutes at 4 °C after 40 minutes of unwinding. DNA damage was analyzed by the comet module of Lucia 6.20 image analysis  after the cells were stained with ethidium bromide . The percentage of DNA in the comet tail was measured. At least fifty cells per slide were analyzed.Whole-cell lysates  were prepared 24 h following treatment of Jurkat and MOLT-4 cells with scoulerine. Cells treated with 0.1% DMSO were used as negative control and cells treated with 5 µM of cisplatin were used as positive control. Quantification of the protein content was performed using the BCA assay . The lysates (20 μg purified protein) were loaded into each lane of a polyacrylamide gel. After electrophoretic separation, the proteins were transferred to a PVDF membrane . Non-specific binding of the membranes was blocked for 1 h in a Tris-buffered saline containing 0.05% Tween 20 (TBS) and 5% non-fat dry milk. The membranes were washed in TBS. Incubation with a primary antibody against specific antigens  was performed at 4 °C overnight. The following day the membranes were washed 5-times with TBS, each time for 5 minutes, and once with TBS, for 10 minutes, and then incubated with an appropriate secondary antibody  for 1 h at room temperature. Band detection was performed using a chemiluminiscence detection kit . To ensure equal protein loading, each membrane was reprobed and β-actin was detected. At least three independent protein evaluations were performed.The descriptive statistics of the results were calculated in Microsoft Office Excel 2010  or GraphPad Prism 5 biostatistics . For quantitative data, normality testing was performed to assess whether parametric or nonparametric tests should be used. The statistics for comet assay results were computed using NCSS software . Differences within the groups were evaluated using the nonparametric Kruskal–Wallis one-way analysis of variance, followed by Dunn’s post-hoc test with Bonferroni’s modification. All experimental data were expressed as median and 25th and 75th percentiles. For other cell culture experiments all the values were expressed as arithmetic means with S.D. of triplicates. For experiments with parametric variables, the significant differences between the groups were analysed using the Student’s t-test.50 values calculations data from viability determined by use of XTT assay were processed by using GraphPad Prism 5 biostatistics  software. Drug concentrations were plotted against cell viability and the IC50 values were determined using non-linear regression.For ICSupplementary Information


Disseminated cryptococcosis is a rare and fatal disease, and limited data exist regarding it in children. This study aimed to investigate the clinical characteristics of disseminated cryptococcosis in previously healthy children in China.Hospitalized patients with disseminated cryptococcosis were enrolled during January 1996 to December 2015 in Beijing Children’s Hospital, Capital Medical University, China. Data on clinical manifestations, laboratory tests, treatment, and prognosis were evaluated.P < 0.05). Lung  was the most commonly invaded organ, but only 9.6% (5/52) of patients had respiratory signs. The most common findings on chest imaging were hilar or mediastinal lymphadenopathy , and nodules , including small nodules in a scattered distribution  or miliary distribution , especially localized in subpleural area. Subsequent invasion occurred in the CNS, abdomen lymph nodes, liver, spleen, peripheral lymph nodes, and skin. In all patients, 42.3% (22/52) and 51.9% (27/52) had elevated eosinophils or IgE, respectively. The positive rate of serum cryptococcal antigen was higher, especially in patients with CNS invasion (approximately 83.3%), than with other primary methods used for pathogen detection, including cerebrospinal fluid (CSF) cryptococcal antigen, cultures of blood, bone marrow, or CSF, and CSF ink staining. The overall mortality rate of pediatric patients in our study was 11.5% (6/52). Some cases had long-term sequela, including hydrocephalus, cirrhosis, or blindness.A total of 52 pediatric patients with no underlying disease were enrolled, including 38 boys and 14 girls. Only 10 cases had a history of exposure to pigeon droppings. Fever, cough, and hepatomegaly were 3 main manifestations of disseminated cryptococcosis. However, headache was more common in patients with central nervous system (CNS) invasion than in patients with non-CNS invasion (Disseminated cryptococcosis can occur in previously healthy or immunocompetent children in China. Lung and CNS were most commonly invaded by this disease. Furthermore, most cases usually showed no obvious or specific symptoms or signs, and therefore pediatricians should pay more careful attention to identify this disease. Cryptococcus belongs to capsulated yeast and can cause an invasive and fatal disease. Most occurs in adults aged 20 to 50 years old, approximately 1%–5% of the population, while in children the incidence is less than 1% . Joshi eWe retrospectively reviewed hospitalized patients less than 18 years old with disseminated cryptococcosis and treated between January 1996 and December 2015 in Beijing Children’s Hospital, Capital Medical University, China. Collected information included the following: (1) demographic features of the patients, history of exposure to pigeon droppings, course of disease and past medical history; (2) clinical features and symptoms; (3) laboratory tests, including full blood cell tests, liver and renal function tests, CD4+ and CD8+ T cells subsets, pathogen tests, imaging, ultrasounds, and pathology; and (4) treatment and prognosis. Information on the prognosis of pediatric patients was collected using telephone follow-up.The diagnostic criteria of disseminated cryptococcosis have been defined as 2 or more non-adjacent organs being simultaneously affected with cryptococcosis . CryptocCryptococcal antigen assay was conducted using the Immy Latex-Crypto Antigens . Blood samples were cultured with chromogenic agar medium (CHRO) and identified by an API-20CAUX strip for yeast-like fungi. Then, we conducted a drug sensitivity test by ATB-FUNGU Strip. These drugs included amphotericin B, fluconazole, itraconazole, and 5-flucytosine.In this study, we defined previously healthy children as patients with no underlying disease, including acquired immune deficiency syndrome (AIDS), prolonged corticosteroid usage, immunodeficiencies, organ transplantation, advanced malignancy, and diabetes. Furthermore, all pediatric patients were with normal numbers of CD4+ T cells, CD8+ T cells, B cells, and NK cells, and had normal levels of IgG, IgA, and IgM.t test. All tests were 2-tailed, and P < 0.05 was considered statistically significant.All data were analyzed using SPSS version 19.0 software. Continuous data are presented as the mean ± standard deviation or range. Proportions were compared by the Chi-squared or Fisher’s exact test, and continuous variables within 2 groups were compared using the independent Fifty-two children with disseminated cryptococcosis were enrolled in this study, 38 of which were male and 14 female, for a male-to-female ratio of 2.7:1. The median age was 4.3 years, ranging from 1.3 years to 11 years. The age distribution of these patients is shown in Table P < 0.05), but lower rate of splenomegaly (P < 0.05) (see Table All 52 patients 100%) had fever (defined as a temperature above 37.3 °C), and the mean peak temperature was 39.3 ± 0.6 °C (range: 38.5–40.5 °C). The incidences of cough, hepatomegaly, and headache were 53.8% (28/52), 51.9% (27/52), and 28.8% (15/52), respectively. Most coughs were mild and dry . Splenomegaly, lymphadenopathy, rash, and other symptoms were also seen in these patients. In addition, 2 patients presented with facial paralysis, 2 with loss of consciousness, and 1 with diarrhea and blindness. Hospitalization time ranged from 2 days to 109 days, with an average of 42.4 ± 27.2 days. Compared with the non-CNS invasion group, pediatric patients in the CNS invasion group had a higher rate of headache  had invasion of 2 organs, while 84.6% (44/52) had invasion of 3 or more organs. Four cases developed invasion of 7 organs (the highest number of invaded organs). In this study, lung  was the most commonly invaded organ, of which 22 patients were without respiratory symptoms, but with fever or headache or other symptoms. The second most commonly invaded organ was the CNS , which manifested with headache, fever, nausea, vomiting, and irritability. Meanwhile, 33.3% (12/36) had no nervous system manifestations but other non-CNS related symptoms. In this study, cranial nerve was invaded in 5 patients (9.6%), including the facial nerve in 2 cases, the oculomotor nerve in 2 cases, and the abducens nerve in 1 case. Cryptococcus was also susceptible to invading the abdomen lymph nodes, liver, spleen, and peripheral lymph nodes, accounting for 67.3%, 63.5%, 50.0%, and 38.5% respectively  white blood counts (WBC) greater than 20 × 109/L. Twenty-two cases (42.3%) had an elevated percentage of eosinophils, ranging from 8.1% to 57% . Meanwhile, decreased hemoglobin, ranging from 63 g/L to 83 g/L, was detected in 26.9% (14/52) of patients. C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) of all patients were increased, and ranged from 11 mg/L to 273 mg/L (average of 86.6 ± 70.9 mg/L), and 28 mm/h to 140 mm/h (average of 52.0 ± 36.3 mm/h), respectively. Sixteen (30.8%) patients’ CRP was higher than 100 mg/L, and 14 (26.9%) patients’ ESR was higher than 100 mm/h. Furthermore, IgE levels increased in 51.9% (27/52) of patients, and the highest was more than 2000 IU/L; however, this decreased quickly to normal levels after anti-fungal treatment. The additional laboratory tests had been performed in patients with high levels of eosinophils and IgE in order to exclude the other causes such as parasitic infection, eosinophilic lung diseases, and hyperimmunoglobulin E syndrome. Levels of IgG, IgA, and IgM were normal, as were the numbers of CD4+ T cells, CD8+ T cells, B cells, and NK cells. The percentage of CD4+ T cells and CD8+ T cells was slightly lower in just 4 cases. Furthermore, HIV was negative in all patients. Lastly, there was no evidence of immunodeficiencies in any patients.Forty-eight patients had leukocytosis with an average of 22.1 ± 8.9 × 10P < 0.05). Cryptococcal antigens in blood decreased slowly, a change that was detectable even 2 years later in 1 patient. In this study, 49 patients underwent lumbar puncture, and the positive of cryptococcal antigens, ink staining, and culture of cryptococcus in CSF samples were 42.9% (21/49), 38.8% (19/49), and 32.4% (11/49), respectively; these values were especially higher in patients with CNS invasion (P < 0.05)  and 46.2% (24/52), respectively, and both were higher in patients with CNS invasion than in patients without CNS invasion (n = 7), cervical lymph node (n = 6), liver (n = 2), and skin (n = 4). Results revealed structural damage, multifocal necrosis, abscesses, granulomatous inflammation, and cryptococcus infiltration. Periodic acid Schiff and methenamine silver staining were positive in all cases , interstitial opacities , and pleural thickening   and nodules , including small nodules in a scatter distribution  or miliary distribution , and especially localized in subpleural area. Other findings displayed patchy shadows , mostly in the right upper lobe  scan, and 44 patients had abnormal results. Hepatomegaly  and splenomegaly  were the most common findings. Among these patients, 15 had parenchymal low-density lesions both in the liver and spleen, while an additional 3 and 5 cases had diffuse nodules in the liver and spleen, respectively  of cases had abnormal neurology imaging. Among them, 52.4% (11/21) had hydrocephalus, including communicating hydrocephaly in 7 cases and non-communicating hydrocephaly in 4 cases. Furthermore, 38.1% (8/21) of cases had ventricular dilatation, 28.6% (6/21) had low-density lesions in the brain parenchyma, 23.8% (5/21) had meningeal enhancement, 14.3% (3/21) had calcification in the brain parenchyma, and 4.8% (1/21) had intracranial venous sinus thrombosis , 5-flucytosine (100 mg/kg/d), and fluconazole (6 mg/kg/d) in the initial treatment. For other treatment strategies, see Table Cryptococcosis is a rare and fatal disease that preferentially infects immunosuppressed hosts. A previous study  reportedIn our study, the median age of children with disseminated cryptococcosis was 4.7 years old, and 63.5% of patients were less than 5 years old. This was similar to Luo et al.’s  study, bIn our study, fever, cough, and hepatomegaly were the 3 most common manifestations, while in the study by Severo et al. , headachBesides lung and brain invasion, Dromer  demonstr9/L), and high levels of CRP and ESR were common in patients with disseminated cryptococcosis. Furthermore, 42.3% and 51.9% of patients had elevated eosinophil and IgE, which is similar to results by Bassetti et al. [In this study, increased WBC  plus 5-flucytosine (100 mg/kg/d) for 2 weeks, followed by fluconazole (6 mg/kg) for a minimum of 8 weeks, and then maintenance therapy with fluconazole for at least 6 to 12 months [In our study, the clinical isolates in Chinese pediatric patients were sensitive to amphotericin B, fluconazole, itraconazole and 5-flucytosine. Chan et al. , 34 foun2 months . Zhu et 2 months  pointed 2 months . However2 months  study de2 months  reportedDisseminated cryptococcosis can be seen in previously healthy or immunocompetent children in China. The lung and CNS were the 2 most commonly invaded organs in this study. Most cases showed no specific clinical manifestations, or were misdiagnosed as tuberculosis or other diseases. The prognosis of children with disseminated cryptococcosis is poor, but if diagnosed early, the majority cases could be managed successfully.


WYC-209 also inhibits proliferation of TRCs of human melanoma, lung cancer, ovarian cancer, and breast cancer in culture. Interestingly, the treated TRCs fail to resume growth even after the drug washout. Importantly, the molecule abrogates 87.5% of lung metastases of melanoma TRCs in immune-competent wild-type C57BL/6 mice at 0.22 mg kg−1 without showing apparent toxicity. Pretreating the melanoma TRCs with retinoic acid receptor (RAR) antagonists or with RAR siRNAs blocks or reduces the inhibitory effect of the molecule, suggesting that the target of the molecule is RAR. WYC-209 induces TRC apoptosis and pretreating the TRCs with caspase 3 inhibitor or depleting caspase 3 with siRNAs substantially rescues growth of TRCs from WYC-209 inhibition, suggesting that WYC-209 induces TRCs apoptosis primarily via the caspase 3 pathway. Our findings demonstrate the promise of the new retinoid WYC-209 in treating malignant melanoma tumors with high efficacy and little toxicity.Developing novel drugs that can abrogate the growth and metastasis of malignant tumors is a major challenge for cancer researchers. Here we describe a novel synthetic retinoid, namely WYC-209, which inhibits proliferation of malignant murine melanoma tumor-repopulating cells (TRCs), known to resist conventional drug treatment, with an IC The discovery of efficient drugs is a main challenge in cancer therapy. Here, the authors report a synthetic retinoid, WYC-209 with anti-tumor and anti-metastatic activity mediated by the retinoic acid receptor signaling pathway. Cancer stem cells (CSCs) or tumor-initiating cells (TICs) are a self-renewing, highly tumorigenic subpopulation of tumor cells. They play a critical role in the initiation and progression of cancer3. CSCs or TICs exhibit high chemo-resistance to conventional chemotherapeutic drug treatment and therefore are speculated to be the key players in cancer relapse after chemotherapy4. As a consequence, developing targeted chemotherapeutic drugs to abrogate CSCs or TICs is a key task in cancer research and clinical application. We have recently developed a mechanical method of selecting and growing tumorigenic cells from cancer cell lines and primary cancer cells by culturing single cancer cells in soft fibrin gels5. The selected cancer cells display high self-renewal ability and are resistant to chemotherapeutic drugs such as cisplatin and doxorubicin5. Remarkably, when injected the selected cancer cells into tail veins, as few as ten of such cells can generate distant metastatic colonization in immune-competent mice. We thus functionally define these soft-fibrin-gel-selected cancer cells as tumor-repopulating cells (TRCs), differentiating them from CSCs or TICs that are selected via cell surface stem cell marker approaches. These TRCs express high levels of self-renewing gene Sox2 and low levels of master differentiation gene Mitf and hence appear to remain undifferentiated or partially differentiated6. Treating TRCs with retinoid acid (RA), which is a nonspecific differentiation factor, could inhibit TRCs extravasation7, a key late stage in metastasis. However, poor water solubility and high toxicity of RA significantly limit its use in clinical treatment of cancer11. In order to develop highly potent retinoids with great efficiency in inhibition of cancer stem cell like TRCs, we have performed in-house drug discovery processes to specifically overcome these limitations. In the current study, we describe synthesis and discovery of a novel retinoid, named WYC-209, which abrogates growth of TRCs of several cancer cell lines in culture and inhibits lung metastasis by melanoma TRCs in vivo, with little toxicity on non-cancerous cells or immune-competent mice.Chemotherapy is one of the principal modes of treatment for cancer, but resistance to chemotherapeutic drugs is a hallmark of malignant tumors that results in major limitation in chemotherapy12. According to the clarified SARs (Structure Activity Relationships) of synthetic retinoids, an aryl carboxylic acid right half which that mimics RA’s terminal acid group is the most important pharmacophore to retinoids13. However, since most retinoids are highly lipophilic compounds with propensity to accumulate in the human body, researchers have identified RA analogs that substitute benzoic acid with aromatic rings; for example, Tazarotene14 4 and (+/−)-DET17   is presumed to function as a tumor suppressor in various contexts since its absence is associated with tumorigenicity and its presence causes cell cycle arrestner Fig.  and the ner Fig. , while aner Fig. . Consistner Fig.  and 33 aner Fig. , suggest24. We cultured B16-F1 or A375 into 3D soft fibrin gels and treated them with the caspase 3 inhibitor z-DEVD-FMK for 3 h before adding WYC-209. Pretreating with z-DEVD-FMK substantially rescued growth of mouse melanoma B16-F1 TRCs  , both WYC-209A and WYC-209B showed negligible hERG channel blocking activity  of these two drugs, suggesting that stem cell-like TRCs might be responsible for drug resistance in malignant melanoma cells. WYC-209 is a new retinoid discovered from the in-house screening of a synthetic retinoid library. Inhibition of B16-F1 TRC growth by WYC-209 is about 10 folds more potent than that of Tazarotene and both enantiomers are highly effective. All-trans retinoic acid is reported to inhibit acute promyelocytic leukemia and breast cancer27 via ablation of Pin1, but toxicity11 and drug resistance29 of the molecule limit its application in human patients. A recent report shows that an MCL1 inhibitor S63845 is effective against several cancers with reasonable tolerance in mice30, but it is not clear at the present time whether this molecule is effective against TRCs. The time-dependent apoptosis of un-selected melanoma cells or melanoma TRCs by WYC-209 reached ~95% 24 h after treatment, much higher than that by Tazarotene  is interesting, since there is no sign of “relapse” or growth of the TRCs in the in vitro cell culture model after single melanoma TRCs were plated back into the soft fibrin gels with fresh medium in the absence of the compound. These results suggest that one might only need to deliver WYC-209 for very limited times to animals or human subjects to abolish the growth potential of malignant melanoma cells. However, whether the long-lasting effect of WYC-209 on melanoma TRCs can be extended to other types of tumorigenic tumor cells in mice or in humans remains to be seen in the future.The binding assay by surface plasmon resonance (SPR) analysis shows that WYC-209A and WYC-209B acid bind to RARs at nano-molar doses, which are similar to their natural counterpart ATRA, and the competition binding assay has demonstrated that the RAR antagonists could rescue TRC growth inhibited by WYC-209. Furthermore, all human cancer cell lines that we have tested expressed RARs and their expression levels are positively correlated with their sensitivity to WYC-209 , ethyl 2-chloropyrimidine-5-carboxylate15 , and Pd(PPh3)2Cl2  was dissolved in DMF (2.0 mL) under argon atmosphere. A mixture of CuI  and triethylamine (0.3 mL) in DMF (1.0 mL) was added. The solution was heated to 75 °C for 22 h. The reaction mixture was cooled to room temperature and filtered on a celite pad, followed by washing with ethyl acetate (30 mL). The filtrate was then washed with brine (20 mL) and dried over anhydrous magnesium sulfate. Evaporation of the solvent under vacuum yielded the crude products, which were purified with flash column chromatography to obtain the pure compound 3 . 1H NMR  δ 9.24 , 7.69 , 7.33 , 7.09 , 4.45 , 3.11–2.99 , 1.99–1.92 , 1.43 , 1.33 ; 13C NMR  δ 163.52, 158.44, 155.85, 142.39, 136.48, 131.36, 130.15, 126.83, 121.88, 115.96, 92.65, 87.97, 62.14, 37.09, 33.14, 30.05, 23.43, 14.38. ESI (+)-MS: 353.2 [M + 1]+; HRMS-ESI (m/z) calculated for C20H21N2O2S, 353.1318 [M + 1]+ and found 353.1317.A mixture of 6-ethynyl-4, 4-dimethylthiochroman3  was dissolved in CH2Cl2 (3.0 mL), and the resulting solution was cooled at 0 °C. m-CPBA  was added drop-wise under stirring and keeping the reaction temperature below 5 °C. After addition, the mixture was stirred for 1 h at 0 °C and 2 h at room temperature. The m-CBA formed was removed by filtration, and the organic phase was washed with a saturated solution of Na2S2O5 (3.0 mL) and with a saturated solution of NaHCO3 (3.0 mL), respectively. The organic phase was dried over anhydrous magnesium sulfate and concentrated, the residue was purified with flash column chromatography to provide WYC-209 . 1H NMR  δ 9.29 , 7.79 , 7.65 , 4.47 , 3.23 , 3.11 , 2.43 , 1.91 , 1.47 , 1.44 , 1.34 ; 13C NMR  δ 163.33, 158.54, 155.32, 145.19, 132.61, 131.06, 130.22, 124.22, 122.62, 89.60, 89.35, 62.32, 43.35, 34.75, 31.36, 31.25, 29.93, 14.39. ESI (+)-MS: 369.4 [M + 1]+; HRMS-ESI (m/z): [M + 1]+ calculated for C20H21N2O3S, 369.1267 and found 369.1267.Compound D-diethyl tartrate  in 1.0 mL of dichloromethane at 16 °C. After 2.5 min, 10 μL of water was added slowly using a microliter syringe with vigorous stirring for 10 min, followed by quickly cooling to −20 °C for an additional 20 min. The reaction was allowed to take place after rapid addition of compound 3  and cumene hydroperoxide . After 3 h the mixture was poured to a solution of citric acid (67 mg) in a mixed solvent of water (4 mL), l, 4-dioxane (2 mL), and diethyl ether (4 mL), and was stirred for 15 min. The aqueous phase was extracted with diethyl ether (3 × 10 mL). The combined organic phases were stirred vigorously with 10 mL of 2 M aqueous K2CO3 for 0.5 h. The aqueous solution was then extracted with diethyl ether (3 × 10 mL). The combined organic solutions were washed with brine (25 mL), dried over MgSO4, and filtered. The enantiomeric excesses (ee) measurement for the enantioselective oxidation was made with the solution, and 75% ee yielded. Then the solution was evaporated under reduced pressure. Flash chromatography of the crude product yielded WYC-209A . The product was recrystallized with ethyl ether for 3 times, and 99% ee product (25 mg) was obtained. All the ee values were measured by chiral HPLC analysis: Agilent 1260 infinity HPLC, Lux Cellulose-1 250 × 4.6 mm column, 20 °C, 254 nm, acetonitrile/H2O = 80:20, 0.7 mL min−1, retention time 7.81 min. The fractions of sulfoxide were mixed before enantiomeric excesses (ee) measurement. [α]D24 =  85.6 ; 1H NMR  δ 9.29 , 7.82–7.75 , 7.66 , 4.47 , 3.24 , 3.15–3.07 , 2.43 , 1.92 , 1.47 , 1.44 , 1.34 ; 13C NMR  δ 163.33, 158.54, 155.32, 145.19, 132.61, 131.06, 130.22, 124.22, 122.62, 89.60, 89.35, 62.32, 34.75, 31.36, 31.25, 29.93, 14.39; ESI (+)-MS 369.4 [M + 1]+; HRMS-ESI (m/z): [M + 1]+ calculated for C20H21N2O3S, 369.1267 and found 369.1267.Titanium tetraisopropoxide  was added rapidly to a solution of L-diethyl tartrate  in 1.0 mL of dichloromethane at 16 °C. After 2.5 min, 10 μL of water was added slowly using a microliter syringe with vigorous stirring for 10 min, followed by quickly cooling to −20 °C for an additional 20 min. The reaction was allowed to take place after rapid addition of compound 3  and cumene hydroperoxide . After 3 h the mixture was poured to the solution of citric acid (67 mg) in 4 mL of water, 2 mL of l, 4-dioxane, and 4 mL of diethyl ether, and was stirred for 15 min. The aqueous phase was extracted with diethyl ether (3 × 10 mL). The combined organic phases were stirred vigorously with 10 mL of 2 M aqueous K2CO3 for 0.5 h. The aqueous solution was then extracted with diethyl ether (3 × 10 mL). The combined organic solutions were washed with brine (25 mL), dried over MgSO4, and filtered. The enantiomeric excesses (ee) measurement for the enantioselective oxidation was made with the solution, and 74% ee yielded. The solution was evaporated under reduced pressure. Flash chromatography of the crude product yielded WYC-209B . The product was recrystallized with ethyl ether for three times, and 99% ee product (23 mg) was obtained. All the ee values were measured by chiral HPLC analysis: Agilent 1260 infinity HPLC, Lux Cellulose-1 250 × 4.6 mm column, 20 °C, 254 nm, acetonitrile/H2O = 80:20, 0.7 mL min−1, retention time 8.22 min. [α]D24 =  -85.5 ; 1H NMR  δ 9.29 , 7.82–7.75 , 7.66 , 4.47 , 3.24 , 3.15–3.07 , 2.43 , 1.92 , 1.47 , 1.44 , 1.34 ; 13C NMR  δ 163.33, 158.54, 155.32, 145.19, 132.61, 131.06, 130.22, 124.22, 122.62, 89.60, 89.35, 62.32, 34.75, 31.36, 31.25, 29.93, 14.39; ESI (+)-MS: 369.4 [M + 1]+; HRMS-ESI (m/z): [M + 1]+ calculated for C20H21N2O3S, 369.1267 and found 369.1267.Titanium tetraisopropoxide  was added rapidly to a solution of Four-week old C57BL/6 female and male mice were obtained from the Center of Medical Experimental Animals of Hubei Province . The mice were randomly assigned to the control group or the treated group. As a minimum of eight mice per group was required for having a statistical power, each group had eight mice. The experimentalists were blinded from the expected outcome of the treatment. All animals received humane care in compliance with the Principles of Laboratory Animal Care Formulated by the National Society of Medical Research and the guide for the US National Institutes of Health. The protocol was approved by the Animal Care and Use Committee of Huazhong University of Science and Technology.2. Cells were passaged every 3–4 days using TrypLE (Life Technologies). Cell samples were randomly allocated to each well of the culture dishes and randomly chosen for intervention. For all cell culture experiments, at least three independent experiments were performed per condition.Murine melanoma cell line B16-F1, human ovarian cancer cell line A2780, human lung cancer cell line A549, human breast cancer cell line MCF-7, human melanoma cell line MDA-MB-435s, human melanoma cell line A375 and non-cancerous murine fibroblast cell line 3T3 were purchased from the China Center for Type Culture Collection , non-cancerous human epidermal cell line HacaT was purchased from Biovector NTCC. In all experiments, cells were randomly allocated to different experimental groups. Since an effective assay for mycoplasma contamination is visual inspection by imaging and DAPI-staining, all cells are counter-stained with DAPI and are constantly monitored for mycoplasma contamination during the course of study. Cells were cultured on rigid dishes with RPMI-1640, Dulbecco’s modified Eagle’s medium or minimum essential medium cell culture medium supplemented with 10% fetal bovine serum , and 1% penicillin and streptomycin at 37 °C with 5% CO6. In brief, fibrinogen was diluted into 2 mg mL−1 with T7 buffer . Cells were detached from 2D rigid dishes and cell density was adjusted to 104 cells per mL. Fibrinogen and cell solution mixture were made by mixing the same volume of fibrinogen solution and cell solution, resulting in 1 mg mL−1 fibrinogen and 5000 cells per mL in the mixture fibrin gels. A volume of 50 μL cell/fibrinogen mixture was seeded into each well of 96-well plate and mixed well with pre-added 1 μL thrombin (100 U mL−1). The cell culture plate was then incubated in 37 °C cell culture incubator for 10 min. Finally, 200 μL of DMEM medium containing 10% FBS and antibiotics was added.Salmon fibrinogen and thrombin were purchased from Reagent Proteins . 3D fibrin gels were prepared as described previously−1 PI and 100 ng mL−1 DAPI  for 1 h. The PI-positive (apoptotic) cells and DAPI-labeled cells were imaged by DMI-6000B fluorescence microscope.Propidium iodide  and 4′, 6-diamidino-2-phenylindole  were used to label apoptotic cells and cell nucleus cultured in 3D fibrin gels on Day 5, respectively. Briefly, the cells cultured in 3D fibrin gels were rinsed with pre-warmed PBS and then incubated with 10 µg mLControl B16-F1, A2780, A549, MCF-7, MDA-MB-435s, and A375 cells were cultured in 90-Pa fibrin gels to form round colonies and then treated with 0.1, 1, and 10 μM WYC-209, WYC-209A, and WYC-209B or 0.1% DMSO on day 3, which was washed out with pre-warmed PBS on day 4 with fresh medium, and then kept culturing till day 7 or day 9.−1 WYC-209, 0.22 mg kg−1 WYC-209, or 0.1% DMSO every two days. The mice were euthanized and examined for lung tumor formation at day 30. In treated TRCs injected in vivo experiments, B16-F1 cells were seeded into 3D 90-Pa fibrin gels and treated with 10 µM WYC-209 or 0.1% DMSO on day 3, then isolated treated TRCs colonies on day 5 and pipetted into single cell. 30,000 of TRCs were intravenously injected into the tail vein of each wild-type C57BL/6 mouse. No blinding was performed in the mice experiments.Four- to six-week-old female and male C57BL/6 mice were used in mice experiment. Mice were randomized into different groups. In metastasis experiment, B16-F1 cell spheroids were selected from 3D 90-Pa fibrin gels and pipetted to single cells. These TRCs were harvested and the cell number was counted under microscopy. The cells were then suspended in PBS with appropriate cell density. Thirty thousand TRCs were intravenously injected into the tail vein of each wild-type C57BL/6 mouse. Five days later, inoculated mice were intravenous implanted with 0.022 mg kgLung, liver, spleen, bone, stomach, and brain of C57BL/6 mice were fixed by 4% paraformaldehyde then were embedded in paraffin and cut to ~4 µm thick sections by Thermo FINESSE 325. Organ sections were stained by H&E and slides were evaluated for tumor formation by a veterinary pathologist blinded to sample identity.−1 Dispase II (Sigma), cells were labeled with FITC-conjugated Annexin-V and PI according to the manufacturer’s instruction (Biolegend). Data were acquired on a BD Cantoll (BD biosciences) and analyzed with FlowJo software. For BrdU staining to quantify cell proliferation, cells were labeled with FITC-conjugated anti-BrdU antibody according to the manufacturer’s instruction  and then data were acquired on an Accuri C6 (BD Biosciences) and analyzed with BD Accuri C6 software.For Annexin-V and PI staining to quantify cell apoptosis, treated TRCs were isolated from 3D fibrin gel by using 5 mg mLTo quantify the expression levels of RARα, RARβ, RARγ, caspase-3, cleaved caspase-3, and cleaved PARP, cells were lysed with RIPA lysis buffer. Each sample were separated by 8–15% SDS-PAGE, blocked with 5% BSA over night at 4 °C, and incubated with primary antibodies to RARα , RARβ , RARγ , caspase-3 , cleaved caspase-3 , or cleaved PARP  overnight at 4 °C. Primary antibodies were detected with mouse anti-Rabbit IgG-HRP , donkey anti-goat IgG-HRP , or anti-Mouse IgG-HRP .DMSO at Day5 − Colony sizeDMSO at Day 0) − (Colony sizeWYC-209 at day5 − Colony sizeWYC-209 at Day0)]/(Colony sizeDMSO at Day5 − Colony sizeDMSO at Day 0).The growth inhibition of human or murine TRCs by WYC-209 were calculated by the formula below: Growth inhibition = 100% × [-2,5-diphenyltetrazolium bromide) colorimetric assay (abcam). Cells were seeded in 3D soft fibrin gel in 96-well microplates at a density to maintain control (untreated) cells in an exponential phase of growth during the entire experiment. Cells were incubated with various concentrations of compounds for each time point followed by incubation with 50 µL MTT reagent (abcam) for 3 h at 37 °C. After incubation, 150 µL MTT solvent (abcam) was added and absorbance was measured at 570 nm. All experiments were repeated at least three times. The percentage of viable cells was calculated and averaged for each well: percent growth = (OD-treated cells/OD control cells) × 100, cell proliferation at each time point is normalized to base line cell survival at the time of initial compound treatment (day 0).−1 with high-performance injection and the data collected at 10 Hz. Both association and dissociation times were 120 s. Prime into run buffer contains PBS with 0.05% P20 and 5% DMSO. Immobilized proteins via the amine coupling procedure were diluted to 50 μg mL−1 in 10 mM sodium acetate . Compound binding was plotted in a 9-point dose–response curve with no compound as the reference. Highest concentration was 10 μM for the compounds. Final DMSO concentration was 5.0% for all compounds. Executed runs included three startup injections and a 4-point solvent correction at the beginning and the end of the run.Surface plasmon resonance (SPR) was carried out by Shanghai Medicilon Inc, using the Biacore 8K equipment. Human protein RARα (ab82196), RARβ (ab82202), and RARγ (ab81922) were purchased from Abcam (US) Ltd. Proteins were immobilized by amine coupling. Experiments were performed at 25 °C at 30 μL minmouse RARα #1; UAAAUGUGCUUAAAAUGAATT for mouse RARα #2; UAUCUGGGGAUUGGUACGCTT for mouse RARβ #1; UAUAUGAACAUAGAAAGCATT for mouse RARβ #2; CUAAUAAAUAAAUAGAGGCTT for mouse RARγ #1; ACUUUGGCAAAAUAACGAGTT for mouse RARγ #2; GGAUAGUGUUUCUAAGGAATT for mouse caspase 3 #1; GCCAACCUCAGAGAGACAUTT for mouse caspase 3 #2; GGGAUCUAUCUGGACAGUATT for mouse caspase 3 #3; CACAGCACCUGGUUAUUAUTT for human caspase 3 #1; CCCUGGACAACAGUUAUAATT for human caspase 3 #2; CCGACAAGCUUGAAUUUAUTT for human caspase 3 #3.Cells were transfected with siRNA using Lipofectamine 2000 (invitrogen) following the manufacturer’s protocol. Silencer Negative Control No. 1 siRNA  was used a negative control in RNA interference experiments. The sequences of siRNAs are: UUAAAAAAAAAAAAUGGUUTT for GoTaq qPCR Master Mix (Promega). The data were normalized against mouse glyceraldehyde 3-phosphate dehydrogenase. The primer sequences are as follows: mouse GAPDH ; mouse RARα ; mouse RARβ ; mouse RARγ ; mouse Sox2 ; mouse Mitf .Total mRNA was isolated from the cells using the Trizol reagent according to the supplier’s instruction (Invitrogen). Reverse transcription (RT) was performed using the TransScript First-strand cDNA Synthesis SuperMix (TransGen), according to the manufacturer’s protocol. Real-time RT-PCR was performed using n ≥ 2 for each dose. The IC50 values were determined with XLFit or Graphpad Prism curve fitting software.The hERG blocking assays for WYC-209A and WYC-209B were evaluated in the CHO cell line stably expressing hERG channels  using automated conventional manual patch clamp , while the cisapride was used as the positive control. Whole-cell tail currents of hERG channels were tested during the experiments at six doses ; S-mephenytoin (35 µM), bufuralol (10 µM), and testosterone (80 µM), respectively, while the control inhibitors for CYP 1A2, 2C9, 2C19, 2D6, and 3A4 were α-naphthoflavone (0.3 µM), sulfaphenazole (10 µM), omeprazole (100 µM), quinidine (2.5 µM), and ketoconazole (2.5 µM), respectively. After incubation for 10, 10, 45, 10, and 5 min, respectively, the metabolic products of these substrates were analyzed by liquid chromatography (LC)-mass spectrometry (MS). All data were obtained from duplicate assays.To evaluate the inhibitive potential of WYC-209A and WYC-209B on human liver microsomes, five representative metabolic enzymes, including cytochromes P450 (CYP) 1A2, 2C9, 2C19, 2D6, and 3A4 were selected and tested with each isomer using well-established specific inhibiting systems for each CYP enzyme. The substrate drugs used for CYP 1A2, 2C9, 2C19, 2D6, and 3A4 were phenacetin (30 µM), diclofenac (10 µM), t-test; when multiple comparisons were carried out within a set of experiments, Bonferroni correction was also performed.Graphpad Prism software was used for generating Kaplan–Meier animal survival plots of 0.1% DMSO and compounds treated mice and performing statistical analysis (using a log-rank test (Mantel–Cox)). Mice lung metastasis experiments were analyzed by Fisher’s exact test. All other experimental data were analyzed using a two-tailed Student’s The X-ray crystal structures of WYC-209A and WYC-209B are stored in Cambridge Crystallographic Data Centre (CCDC) with the accession codes CCDC 1822478 and CCDC 1822479. Other data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary Information


B-peru gene encoding a basic helix loop helix (bHLH) transcription factor (TF) together with the mPAP1 gene encoding an R2R3 MYB TF to enhance flower color in tobacco (Nicotiana tabacum L.), using the tobacco anthocyanidin synthase (ANS) promoter (PANS) to drive flower-specific expression. The transgenic tobacco plants grew normally and produced either dark pink (PANSBP_DP) or dark red (PANSBP_DR) flowers. Quantitative real time polymerase chain reaction (qPCR) revealed that the expression of five structural genes in the flavonoid biosynthetic pathway increased significantly in both PANSBP_DP and PANSBP_DR lines, compared with the non-transformed (NT) control. Interestingly, the expression of two regulatory genes constituting the active MYB-bHLH-WD40 repeat (WDR) (MBW) complex decreased significantly in the PANSBP_DR plants but not in the PANSBP_DP plants. Total flavonol and anthocyanin abundance correlated with flower color, with an increase of 1.6–43.2 fold in the PANSBP_DP plants and 2.0–124.2 fold in the PANSBP_DR plants. Our results indicate that combinatorial expression of B-peru and mPAP1 genes under control of the ANS promoter can be a useful strategy for intensifying flower color without growth retardation.Flower color is a main target for flower breeding. A transgenic approach for flower color modification requires a transgene and a flower-specific promoter. Here, we expressed the  Rosa hybrid), cyclamen (Cyclamen persicum) and chrysanthemum (Chrysanthemum morifolium) + in the tobacco petals. Moreover, the trace level of delphinidin derivative showing a m/z 611 [M]+ that can be assumed to be delphinidin-3-O-rutinoside was detected in the PANSBP-DR line  and the level of delphinidin derivative in PANSBP_DR lines was 0.06 ± 0.13 mg∙g−1  analysis. The major anthocyanin in the pink flowers of tobacco is known to be cyanidin-3-e (Cy3R) . Our res-DR line A,B. The 3 mg∙g−1 C. SimilaVvMYB5a, VvMYB5b and VvMYBA1 genes reduces the pH in petals from transgenic petunia [ANTHOCYANIN1 (AN1), AN2, and AN11 in petunia increases the pH in petal extracts [B-peru and mPAP1 , some DFRs exhibit different substrate specificity, resulting in species-specific anthocyanin pigments accumulation. Petunia’s DFR does not prefer DHK as a substrate and thus produces almost no pelargonidin-based anthocyanin , while Dduce DHK . In the ynthesis . TTG1, which encodes a WDR protein, displays a similar expression pattern between anthocyanin-accumulating and acyanic tissues [NtTTG1 decreased significantly in both the PANSBP_DP and PANSBP_DR transgenic lines, the petals of which displayed large amounts of anthocyanin , digested with HindIII and SpeI, and then ligated into similarly digested pBB. To construct the final vector for flower-specific expression of both genes (pPANSBP), the PANS::mPAP1::T35S fragment was amplified with PANS-HindIII-F/T35S-R1 primers, and then PANS::B-peru::T35S and the PANS::mPAP1::T35S fragments were digested with HindIII and ligated together. The resulting vector, pPANSBP  plants were generated by transforming tobacco with Agrobacterium containing the pPANSBP construct with the leaf disc method [3 lines were developed by successive self-pollination of T0 plants. The flower phenotypes were checked at three flower developmental stages, S1, S2, and S3.Transgenic tobacco  and two chromatic components a* and b* of the CIE L*a*b* color coordinate  using a Relative changes in cell sap pH (correlating to changes in vacuolar pH) were determined by extracting 0.5 g of macerated petals in 5 mL of bidistilled water. After 2 h of occasional stirring, the pH of the solution was measured with a pH meter . Each measurement was repeated with five individual flowers for each developmental stage.NtF3′5′H, NtMYB3 and NtETC1 are listed in Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, which was previously used in gene expression analysis of transgenic tobaccos was used as an internal reference [Total RNA from tobacco roots, stems, leaves, floral tissues including sepals, petals, stamens and pistils were prepared using TRIzol Reagent (Invitrogen) and first-strand cDNAs were generated using the cDNA EcoDry kit . The qPCR conditions and gene-specific primers are described in previous studies ,30. Geneeference ,30. Threg for 10 min at 4 °C the supernatant was transferred to a new tube. The remaining pellet was rinsed twice with 50 μL of 50% methanol containing 1.2 N HCl, and each extract was combined and subjected to HPLC. HPLC was performed on an LC-20A HPLC system  equipped with an Inertsil-ODS3 C18 column . The mobile phase was composed of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The gradient profile was optimized as follows: 0–30 min, linear gradient 5–55% (v/v) B; 30–45 min, linear gradient 55–65% (v/v) B; 45–50 min, linear gradient 65–100% (v/v) B at a flow rate of 1 mL∙min−1. The temperature of the column compartment was maintained at 40 °C. A diode-array detector was used for compound detection. The spectra of the compounds were recorded between 210 and 800 nm and the peak corresponding to each compound was identified by comparing the retention times and UV spectra with those of the standards. To analyze flavonoid aglycones, acid hydrolysis was carried out. Flower samples at S3 stage were harvested and disrupted in liquid nitrogen. Then, 50 mg of each sample was mixed with 150 μL of 50% methanol containing 1.2N HCl and incubated at 80 °C for 2 h. After centrifugation at 15,000× v/v/v) and then incubated at room temperature for 1 h with shaking. After centrifugation at 15,000× g for 10 min at 4 °C, the supernatant was transferred to a new tube. Additional extractions with 300 μL of MFW solution for 10 min were repeated twice for each sample, and each extract solution was combined with the first extract. Extracts were filtered through 0.45 μm Teflon polytetrafluoroethylene syringe filters, diluted 10-fold in MFW solution, and then subjected to UPLC-QTOF/MS analysis. The analysis was conducted on UPLC-DAD-ESI-QTOF/MS system . The separation was performed with a Luna Omega 1.6 μm column  , a SecurityGuardTM ULTRA cartridges C18 for 2.1 ID pre-column (Phenomenex), and was operated at a temperature of 35 °C. The mobile phase consisted of 0.5% formic acid in water (A) and 0.5% formic acid in acetonitrile (B) at a flow of 0.3 mL∙min−1 using the following gradient program: 0–2 min, isocratic 7% (v/v) B; 2–24 min, linear gradient 7–15% (v/v) B; 24–40 min, linear gradient 15–30% (v/v) B; 40–48 min, linear gradient 30–60% (v/v) B; 48–50 min, isocratic 60% (v/v) B; 50–53 min, linear gradient 60–90% (v/v) B; 53–54 min, isocratic 90% (v/v) B; 54–55 min, linear gradient 90–7% (v/v) B; 55–60 min, isocratic 7% (v/v) B. Specific wavelengths were monitored separately at 288 nm for flavanones and dihydroflavonols, and 350 nm for flavones and flavonols, and 520 nm for anthocyanins. The UPLC was coupled to a Xevo G2-S QTOF-ESI/MS (Waters MS Technologies). The scan range was m/z 50–800 in positive ion mode. The capillary and sampling cone voltages were 3.5 kV and 40 V, respectively. The desolvation gas was maintained at 1050 L∙h−1 at a temperature of 500 °C. The cone gas was maintained at 50 L∙h−1 with an ion source temperature of 120 °C. Data acquisition and processing was performed using MassLynx version 4.1 software (Waters MS Technologies).To identify individual anthocyanin glycosides, UPLC-QTOF/MS analysis was conducted. The ground sample (0.05 g) was mixed with 600 μL of MFW solution (methanol:formic acid:water; 50:5:45;


Houttuynia cordata Thunb (HC) is a traditional herbal medicine widely used in Asia for the treatment of patients with alopecia, usually in combination with other two herbal medicines (Perilla frutescens var. acuta (PFVA) and green tea (GT)). However, the effect of this herbal complex has not been clearly demonstrated. We sought to determine the hair growth-promoting effect of this herbal complex  in the animal model.β1 was performed.Six-week-old male C57BL/6 mice were randomly divided into four groups  as a positive control, and two (200 and 400 mg/kg) concentrations of the herbal complex as experimental groups) and were fed its corresponding medications orally for 25 days. Hair growth was evaluated visually and microscopically. Western blot analysis for insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-β1 was 0.26 (±0.01), 0.19 (±0.02) and 0.15 (±0.01) in negative control, the 200 and 400 mg/kg group, respectively.The herbal complex exhibited hair growth-promoting activity in C57BL/6 mice. Grossly, the area of hair regrowth was 55.1 (±3.8) %, 70.2 (±6.3) % and 83.5 (±5.7) % in negative control, herbal complex 200 mg/kg and 400 mg/kg group, respectively. In histologic examination, the hair follicle count in deep subcutis was 2.6 (±0.7), 5.8 (±0.7) and 8.6 (±1.2) and the diameter of hair follicles was 11.9 (±5.0) μm, 17.4 (±3.9) μm and 22.8 (±5.2) μm in negative control, herbal complex 200 and 400 mg/kg group, respectively. The expression of IGF-1 was 0.14 (±0.01), 0.23 (±0.02) and 0.24 (±0.01) and the expression of TGF-This data provides adequate preliminary experimental evidence to support the hair regeneration effect of this herbal complex.The online version of this article (10.1186/s12906-017-2003-x) contains supplementary material, which is available to authorized users. The boundary between black and gray areas of skin was defined subjectively, by a single doctor (MSC). The measurements were done blinded to treatment. The skin was then isolated to examine the histological features after the sacrifice of all mice at day 25. Individual skin samples were fixed in 10% paraformaldehyde , and the tissues were then dehydrated using alcohol and xylene and embedded in paraffin using an automatic tissue processor, sectioned to 4 μm thickness with a microtome, and stained with hematoxylin and eosin. Then, histological analyses were performed using light microscopy . Using a digital photomicrograph, we counted the number of hair follicles and all of the images were cropped in a fixed area (150 pixels in width). Data were evaluated from representative areas at a fixed magnification of 100×. Hair follicle mean diameter, distance between hair follicle and dermis, and length of hair follicle were measured under the microscope.β1 (an inhibitor of hair growth) in the four groups of experimental mice. The levels of IGF-1 and TGF-β1 protein in skin were measured by Western blotting. Fifty milligrams of tissue with lysis buffer  was homogenized  and the homogenized samples (50 μg of protein) were subjected to 15% SDS-PAGE and then transferred to a PVDF membrane. Membranes were blocked in 5% skim milk for 1 h, incubated with primary antibodies  overnight (4 °C) at appropriate dilutions (1:1000), and then incubated with the secondary antibody  conjugates for 1 h at room temperature. Blots were developed using a commercial enhanced chemiluminescence system, and densitometric analysis was performed using the Image Analyzer system .We analyzed the expression of insulin-like growth factor (IGF)-1 (a stimulator of hair growth) and transforming growth factor (TGF)-p < 0.05 was considered statistically significant.Results are presented as mean ± standard deviation (SD). Data were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple comparison test. We used SPSS, version 18  for all statistical analyses. A value of The yield of the obtained HC, PFVA and GT as calculated from dried raw material was 13%, 9% and 11%, respectively. From the HPLC chromatogram, the contents of quercetin, RA and tannin were determined as 0.14 ± 0.08 mg/g of HC, 17.7 ± 0.80 mg/g of PFVA and 56.74 ± 0.17 mg/g of GT, respectively. The HPLC profile of HC, PFVA and GT  is shown in Fig. p < 0.001) was noted in all experimental groups and in the finasteride group, relative to the negative control group  and the positive control finasteride group showed darker skin than the negative control group Fig. . When weoup Fig. . Higher oup Fig. .Fig. 2Grp < 0.05; Table As shown in Fig. p < 0.05). The expression of IGF-1 was increased by 64% and 70% in experimental groups compared to negative control . TGF-β1 expression was lower in all experimental groups and the positive control group compared with the negative control group (p < 0.05). The expression of TGF-β1 was decreased by 24 and 44% in experimental groups  compared to negative control group. The higher doses (400 mg/kg) used in the experimental group resulted in lower expression of TGF-β1 (p < 0.05) relative to the low dose (200 mg/kg) groups  exhibited hair growth-promoting activity based on analysis of gross morphology, histological finding, and expression of IGF-1 and TGF-β1, which plays an important role in the regulation of differentiation, proliferation, and apoptosis in various types of cells, is a possible negative regulator of hair follicle growth and is highly expressed during the catagen and telogen phases [β1 are considered to stimulate and inhibit hair growth, respectively.IGF-1 has been reported to increase the growth of cultured hair follicles. Numerous studies have reported that IGF-1 enhances follicular proliferation, tissue remodeling, and the hair growth cycle, as well as follicular differentiation –44. In an phases . Consequβ1: we found a report that procyanidin B-2, which is one of the principle element of HC [β1 (leading to conversion of telogen follicles to anagen hair follicles) [2 in procyanidin B group vs. 0.08/0.25 cm2 in placebo group, after sequential use for 6 months) in male patients [β1, resulting in the induction of anagen hair regrowth. Consequently, we analyzed the expression of IGF-1 and TGF-β1 after HC-containing herbal medicine treatment. As shown in Fig. p < 0.05) and TGF-β1 expression was much lower (p < 0.05) in the experimental group (both the 200 and 400 mg/kg groups) compared with the negative control group (The expression of IGF-1 was 0.14 (±0.01), 0.23 (±0.02) and 0.24 (±0.01) and the expression of TGF-β1 was 0.26 (±0.01), 0.19 (±0.02) and 0.15 (±0.01) in negative control, the 200 and 400 mg/kg group, respectively). We think our results may suggest the mechanism of action of HC with respect to hair regeneration.Regarding the undiscovered mechanism of action of the hair promoting effect of HC, we believed that HC has a certain element that modulates IGF-1 and TGF-nt of HC , 15, canllicles) . In 2000patients . AccordiHPLC–UV chromatogram in current study showed that quercetin was contained in HC. Quercetin is one of the most abundantly distributed flavonoids found in various food sources such as apples, cranberries, green leafy vegetables, seeds, flowers, olive oil, etc. . This flRosmarinus officinalis L.), spearmint (Mentha spp.), and lemon balm , and also in plants used in traditional Chinese medicine, such as Perilla frutescens (L.) Britton, Salvia miltiorrhiza Bunge, and Rabdosia rubescens (Hemsl.) H. Hara [Perilla frutescens demonstrated effective hair growth regeneration potential [RA is a polyphenolic phytochemical found in several medicinal plants of the Lamiaceae family, such as rosemary  showed higher levels of expression of IGF-1, although this difference was not statistically significant. Similarly, the experimental group (400 mg/kg group) showed lower levels of TGF-Throughout the 25-day period of observation, no fatalities occurred in any of the mice group. All mice appeared to be active and well-groomed before euthanasia. No significant weight loss was noted  by HC, (2) anti-inflammatory and anti-microbial actions [α-reductase by GT [Nevertheless, the present study provides adequate preliminary data to establish a mechanism of action for this herbal complex with respect to hair regeneration. Here, we hypothesize that the multi-herbal formulation achieves its bioactivity with possible synergistic effects from each component: (1) most importantly, induction of anagen hair regrowth , which is commercially available and popularly used in Korea, despite the lack of reasonable experimental evidence of its effectiveness for hair regeneration. Overall, based on our analysis of gross morphology, histological finding, and expression of IGF-1 and TGF-Additional file 1: Table S1.Table S2. Raw data of body weight (g) of C57BL/6 mice. Table S3. Raw data of gross area of hair regrowth (%) at day 25. Table S4. Raw data of diameter (μm) of hair follicle. Table S5. Raw data of length (μm) of hair follicle. Table S6. Raw data of distance (μm) between hair bulb and dermis. Table S7. Raw data of number of hair follicles in deep subcutis. Table S8. Raw data of western blot assay (IGF-1). Table S9. Raw data of western blot assay (TGF-β1) (ZIP 77 kb)Change in body weight (g) of C57BL/6 mice during study period.