Lung cancer remains the leading cause of cancer-related death worldwide, with one-third diagnosed with locally advanced (stage III) disease. Preoperative induction chemo-radiotherapy is key for the treatment of these patients, however conventional cisplatin based approaches has apparently reached a plateau of effectiveness. In the search for new therapies, the targeting of tumor metabolism is revealed as an interesting option to improve the patient’s responses. Here we describe the importance of PGC-1alpha and GAPDH/MT-CO1 ratio levels as surrogates of the Warburg effect from a series of 28 stage III NSCLC patients, on PFS, OS and PET uptake. Moreover, our results show a great variability between tumors of different individuals, ranging from very glycolytic to more OXPHOS-dependent tumors, which compromises the success of therapies directed to metabolism. In this sense, using 3 different cell lines, we describe the relevance of Warburg effect on the response to metabolism-targeted therapies. Specifically, we show that the inhibitory effect of metformin on cell viability depends on cell’s dependence on the OXPHOS system. The results on cell lines, together with the results of PGC-1alpha and GAPDH/MT-CO1 as biomarkers on patient’s biopsies, would point out what type of patients would benefit more from the use of these drugs. Histologically, we can differentiate two types: small-cell lung cancer and non-small cell lung cancer (NSCLC), being the latest the most common, counting for the 80–85% of the cases4. Thus, preoperative induction chemotherapy is key, because it can early eradicate distant micro-metastases and the down-staging increases the number of patients whose tumors are considered resectable, which contributes to improve survival5. After chemotherapy, surgery is potentially curative for these patients, being offered to the vast majority of patients with resectable N2 and to some patients with N3 node involvement (TNM staging system).Approximately one-third of patients with NSCLC are diagnosed with locally advanced (stage III) disease. Currently, the therapeutic strategy for these patients is the neo-adjuvant multimodal treatment, since local therapy alone (surgery or radiation) leads to poor overall survival because most of the patients die of distant metastases6. Over the last few years, based on genetic alterations present in the tumor, new targeted therapies focused on the inhibition of key pathways for tumor proliferation have started to appear. However, these therapies do not cover most patients and have not yet reached the clinical routine for stage III patients. Thus, a need for new effective chemotherapy regimens that can be combined with local therapies (surgery and/or radiation) is still open7. In this sense, there is a considerable interest in identifying novel targeted agents that interfere with other hallmarks of NSCLC, such as tumor metabolism, which has recently aroused great interest.Sadly, conventional treatment of NSCLC has apparently reached a plateau of effectiveness in improving survival of patients, and treatment outcomes must still be considered disappointing8. This predilection of glycolysis over the complete oxidation of nutrients via oxidative phosphorylation (OXPHOS) in the presence of oxygen (such as that performed by differentiated tissues), has to do with the facilitation to incorporate nutrients into biosynthetic pathways that support a rapid cell proliferation9.The Warburg effect describes the preference of tumor tissues to obtain energy through glycolysis regardless of oxygen availability12. However, despite its importance, the PGC-1alpha role in glucose metabolism reprograming in NSCLC has not been studied14.PGC-1alpha is a transcriptional coactivator responsible for mitochondrial biogenesis. Thus in general terms, high levels of PGC-1alpha are associated with more OXPHOS-dependent metabolism of the cells, whereas low levels are associated with a more glycolytic phenotypein vitro and in vivo of its possible clinical utility16. In fact, several clinical trials based on this have been done or are currently ongoing, although their clinical benefits have not been as successful as one might expect17.Since the Warburg effect was described, many drugs targeting cancer metabolism have been developed. Unfortunately, there is a current unavailability on cancer drugs in the clinic targeting the tumor metabolism for lung cancer, despite the existence of scientific evidence Remarkably, at the moment there are no good markers of response, most probably because mechanisms at the molecular level are still not clear. Thus, the characterization of these biomarkers is fundamental in order to identify patients who can benefit more from certain drugs targeting metabolism. This could explain why the clinical trials carried out have not been successful.Therefore, knowing that the fundamental basis of treatment and possible cure of these patients is the neoadjuvant chemotherapy, we set out to identify the role of glucose metabolism in the stratification of patients at this stage, and in a second phase, to link these molecular findings with the sensitivity to different metabolic treatments.To evaluate the importance of Warburg effect on NSCLC patients, we first analyzed the prognostic value of PGC-1alpha and GAPDH/MT-CO1 ratio mRNA levels on Overall-Survival (OS) and Progression Free Survival (PFS). In order to select a group of patients with similar characteristics, we focused on a cohort of 28 stage IIIA NSCLC patients. In this cohort the median PFS and OS were 18 [95% CI: 10.1–26.1] and 22.2 [95% CI: 8.9–35.4] months respectively.PGC-1alpha is responsible for mitochondrial biogenesis. On the other hand, the GAPDH/MT-CO1 ratio is indicative of the balance between glycolysis and OXPHOS metabolism. GAPDH  is a key enzyme in glycolysis and MT-CO1 is one of the three mitochondrially encoded subunits that form the core of the Complex IV of the OXPHOS system.Our results reveal a decreased OS for patients with PGC-1alpha low levels, median 15,4 months [95% CI: 7–23 months], compared to high levels patients with an OS superior to 24 months Fig. .Figure 1With respect to the GAPDH/MT-CO1 ratio, the patients were grouped into tertiles, grouping the medium and high levels in the same category since they behaved similarly compared to the low tertile. The median OS for low levels was higher than 24 months while for medium or high levels were 22.2 and 17.9 respectively (data not shown). Opposed to PGC-1alpha, low levels of the GAPDH/MT-CO1 ratio are indicative of a higher OS compared to the medium-high expression group: low ratio OS >24 months versus medium-high ratio 19.4 months [95% CI: 11.3–27.4 months] Fig. .Although studies on PFS were not statistically significant, a clear difference was observed in the same sense as for OS, being high PGC-1alpha levels and low GAPDH/MT-CO1 ratio good prognostic factors. Comparing the PFS medians for both markers these differences are clearer: low PGC-1alpha 8,5 months [95% CI: 6.7–10.2] vs high PGC-1alpha 22 months [95% CI: 15.6–28.4], and medium-high GAPDH/MT-CO1 ratio 11,9 months [95% CI: 7.4–16.3] vs low GAPDH/MT-CO1 ratio 22 months. These obvious differences in median PFS are lost with the disease course, as the disease progresses in all patients Fig. .These results clearly indicate that a more glycolytic metabolism  confers a worse prognosis in both PFS and OS. Furthermore, the expression levels of both variables correlate inversely with a negative correlation index of 61% Fig. .On the other hand, from the diagnostic positron-emission tomography (PET) images of the patients, the maximum standardized uptake value (SUVmax) levels of the tumor lesions were calculated. Due to the low number of PET images, the cohort was expanded to include other stages, thus obtaining a total of 22 patients with SUVmax information. The results show an increase in SUVmax levels in patients with medium-high levels of GAPDH/MT-CO1 Fig. .Finally, to directly assess the role of the Warburg effect on the proliferative capacity of tumor cells, we analyzed the number of mitosis in hematoxylin-eosin tumor sections and correlated them with the expression levels of PGC-1alpha and GAPDH/MT-CO1 ratio as metabolism biomarkers. These data demonstrate an inverse correlation of the number of mitosis with PGC-1alpha levels (correlation of 61%) and a direct correlation with GAPDH/MT-CO1 ratio levels (correlation of 45%) Fig. .0 cell line, derived from the A549 cell line, lacks the mitochondrial DNA (mtDNA) and serves as negative control of the OXPHOS function.To understand the molecular role of the Warburg effect in NSCLC, we decided to move on to an in vitro model using established cell lines. Three cell lines  from different origins and with different mutational profiles were chosen Fig. . The A54First, we set out to determine whether these cell lines have different aggressiveness as a consequence of their different origins: A549 primary tumor, H1299 lymph node metastasis and H460 pleural effusion. For that we characterized their migration capacity through wound healing assays and their clonogenicity. Our results showed an increased clonogenicity for H1299 and H460 cell lines compared to A549 cell line Fig. . Moreove0 line (without OXPHOS function) is unable to grow with galactose as sugar.Next, as a first approach to define their glucose metabolism, namely more glycolytic- or OXPHOS-orientated, we compared the cells abilities to grow in the presence of either glucose (Glc) or galactose  as carbon source Fig. . Our resThen, to study further in-depth their metabolism and to verify that they behave in a similar way to the patients , we analyzed different parameters such as oxygen consumption, reactive oxygen species (ROS) levels, glucose consumption and PGC-1alpha and GAPDH/MT-CO1 ratio levels.Consistent with previous findings, the oxygen consumption rate showed a similar behavior to the Gal/Glc ratio, with significant differences between the H460 and A549 or H1299 cell lines. Despite the decrease in oxygen consumption of H1299 cells compared to the A549 cells, this difference does not become statistically significant Fig. .Figure 3ROS levels are associated with defects in the OXPHOS function and with a higher degree of tumor progression. Our results indicate that H1299 has the highest ROS levels among the cell lines, followed by H460 cells which have higher ROS levels than A549 cells Fig. .Glucose consumption was assessed with the 2-NBDG fluorescent probe, similar to the fluorodeoxyglucose (FDG) used in patient’s PET images. We observed a significant increase in glucose consumption for the H1299 and H460 cell lines with a more glycolytic profile, although no differences were observed between them Fig. .Finally, PGC-1alpha and GAPDH/MT-CO1 levels were evaluated Fig. . A549 ceOnce the cell lines were metabolically characterized and verified that the relationships between metabolism and aggressiveness parameters were similar to those found in patients, we aimed, as a proof of concept, to test their sensitivities to two drugs (2-deoxy-glucose and metformin) targeting different types of metabolism.It has been reported that metformin acts as an inhibitor of Complex I of the OXPHOS system. To check the effect of metformin on the OXPHOS function, we treated all cell lines with 1 mM or 10 mM metformin for 24 hours Fig. .Figure 40, whose MIMP remained constant with the treatments.Our results showed a significant decrease in mitochondrial inner membrane potential (MIMP) for all cell lines dependent on the concentration of metformin present. The only cell line that did not present this effect was the A549ρ0 are not sensitive to the effect of metformin at low doses since there are no significant differences compared to untreated cells. Consistent with this result, the clonogenicity assays showed greater metformin sensitivity for A549 than for H1299 and H460 cell lines, which exhibited reduced metformin sensitivity , showing a decrease in the number of viable cells for A549 cell line for both doses in a concentration dependent way. On the contrary, it appears that cell lines H1299, H460 and A549ρity Fig. .0 cell line is extremely sensitive (there are no viable cells after treatment), followed by H1299 and H460 cell lines which reduce their viability to approximately 50%. Finally, the A549 cell line is the least sensitive, reducing its viability only 20% compared to untreated cells. In the same way, the number of colonies after 2-deoxy-glucose exposure is lower for H1299 and H460 compared to A549 cells  and tumors with an energetic profile more based on their OXPHOS system . In addition, our results confirm that the Warburg effect has a clinical relevance in NSCLC, both in PFS and OS. The majority of the studies in stage IIIA, even with the heterogeneity of these patients, show a PFS and OS in the order of months, which vary in function of diverse prognostic factors. In our cohort of patients the median OS was 22 months similar to other stage IIIA patient cohorts19. The inverse relationship of PGC-1alpha and GAPDH/MT-CO1 ratio with PFS and OS of patients coincides with the function of these genes related to glucose metabolism and with the Warburg effect. Indeed, both variables correlate inversely, reinforcing this affirmation.Thereby, our results demonstrate that the glycolytic phenotype correlates with a worse prognosis in both PFS and OS in stage IIIA NSCLC patients. Similar conclusions for stage I NSCLC patients were obtained from the proteomic-based bioenergetic cellular indexin vitro studies with cellular models to find markers that define sensitivity to drugs directed to glucose metabolism.Next, due to the impossibility of directly asses drug response in patients, we performed 20.Prior to determine their drug sensitivity, we characterized their aggressiveness and biochemical function. Similar to the results in patients, our cell lines with different aggressiveness showed different metabolisms. A549 cell line showed the weakest malignant phenotype, with low clonogenic potential, migration capacity and a growth rate. On the other hand, H1299 and H460 showed higher aggressiveness than A549 cell line for most of the analyzed parameters. Regarding to metabolism characterization, A549 cell line showed a more OXPHOS metabolism, with higher Gal/Glc index, greater oxygen consumption, lower glucose consumption, elevated levels of PGC-1alpha and lower levels of GAPDH/MT-CO1, whereas the tumor cell lines H1299 and H460 turned out to be more glycolytic, with lower Gal/Glc index, lower oxygen consumption, greater consumption of glucose, reduced levels of PGC-1alpha and elevated GAPDH/MT-CO1 ratio. Nevertheless, there are differences between H1299 and H460 cell lines for some of these parameters although not enough clear to observe a pattern, which make it impossible to conclude differences between these two cell lines. However, the relationship between higher glycolysis with increased malignancy described in our cell lines is similar to other tumor types21. In our study we demonstrate that this compound reduces the MIMP of cells, most probably as a consequence of Complex I inhibition, since Complex I is mainly responsible for MIMP maintenance. Thus, treatment with metformin does not reduce MIMP in A549ρ0 cells, since it is maintained by another mechanism and not through Complex I activity22. Furthermore, when A549 cell line (the one with higher OXPHOS function and highest metformin sensitivity) lacks the mtDNA and therefore is forced to lose its OXPHOS dependence, is no longer sensitive to metformin. On the other hand, H1299 and H460 cell viability is not severely affected, even though metformin decreases their MIMP. This is probably because their MIMP and the associated OXPHOS function are not so important for their growth, as demonstrated by their biochemical parameters. In contrast, 2-deoxy-glucose, which inhibits hexokinase, and blocks the glycolytic pathway, affects the cell lines that are more glycolysis-dependent, such as H1299, H460 and A549ρ0 but does not disturb the more OXPHOS-dependent A549 cell line.Finally, we measured cell lines’ sensitivity to metformin and 2-deoxy-glucose. We propose that metformin affects the cells more when they are more OXPHOS-dependent. Recently, it has been described that metformin is an inhibitor of the complex IOur study point out, through PGC-1alpha levels, an easy manner of assessing the metabolic phenotype in patients and also suggest a correlation with sensitivity to metabolism targeted drugs. The approach proposed, focused more on the phenotype than on the tumor genotype, avoids the difficult interpretation of complex genomic results, even more for a phenotype that can be regulated by multiple genes, as well as, by epigenetic and microenvironmental factors. Further studies will clarify the relationship of these factors in lung cancer, defining the tumor metabolism in a more comprehensive fashion. This knowledge could have implications for the patients’ diagnosis, follow-up and treatment.in vitro drug-sensitivity in cell lines would suggest that PGC-1alpha and GAPDH/MT-CO1 levels could be used to predict patient response to treatments based on targeting glucose metabolism on lung cancer.To summarize, our results indicate that exist variability in glucose metabolism pathways in lung cancer patients’ tumors and established cell lines. In this respect, PGC-1alpha mRNA levels inversely correlate with GAPDH/MT-CO1 ratio. Interestingly, both markers have a clinical relevance in prognosis (PFS and OS), supporting the relationship between a more glycolytic metabolism with a more aggressive phenotype. Furthermore, the All the experiments carried out in this study complied with current Spanish and European Union laws and the principles outlined in the Declaration of Helsinki. The study and experimental protocols were approved by the Hospital Universitario Puerta de Hierro Ethics Committee and written informed consent was obtained for all the patients recruited.A cohort of 28 stage IIIA NSCLC patients was studied. Clinical data from these patients: Overall-survival (OS), Progression-Free survival (PFS), SUVmax levels, etc. as well as molecular data from diagnostic biopsies, were obtained for the analysis.Unfortunately we were only able to obtain PET images for 8 of these patients. Thus, the cohort was increased for SUVmax correlations, retrieving the SUVmax levels from another 17 patients to a total number of 25 patients with SUVmax data .23. Per sample, 10 HPF were analyzed by two independent anatomical pathology technicians and the mean was calculated.The number of mitotic figures was evaluated in the hematoxylin-eosin diagnostic slides using high-power field (HPF) at × 400 magnification0 cells were kindly provided by Dr. Rafael Garesse, from UAM (Madrid). All cells were cultured routinely in DMEM, with 10% Fetal Bovine Serum (FBS), penicillin streptomycin (Gibco) and uridine (Sigma). The mutational landscape of TP53, KRAS, NRAS, PIK3CA, LKB1 was retrieved from COSMIC database.A549, H1299 and H460 were obtained from the ATCC. A549 ρCells were cultured in a 24-well plate until they reached 90% confluence and then scratched with a 200 µL pipette tip. The wounded area was photographed at the start (t = 0 h) and at time point (t = 16 h). Pictures were analyzed using Image J software and the migration rate was calculated as the distance recovered by the migration front per time unit (µm/h). Assays were performed in at least 3 independent experiments.The RNA from cells was extracted using the “RNeasy mini Kit with DNAse” (Qiagen). The RNA extraction from FFPET biopsies was performed using the “High Pure FFPET RNA isolation Kit” (Roche). cDNA was synthetized using the “NZY First-Strand cDNA Synthesis Kit” (NZYtech).24.mRNA expression was evaluated by qRT-PCR using the following Taqman® gene expression assays: PGC-1alpha(Hs01016719_m1), GAPDH (Hs02758991_g1), MT-CO1 (Hs02596864_g1). TBP (Hs00427621_m1) was used as endogenous controlDoubling Time Software v1.0.10 (http://www.doubling-time.com). Assays were performed by duplicate in at least 3 independent experiments.Cell growth was evaluated after seeding 6 well plates at a density of 25,000 cells/well and growing the cells for 4 days in DMEM containing either 4.5 g/L glucose (Glc) or 0.9 g/L galactose . The cells were harvested and counted every 24 hours. Population doubling times were calculated using 3 cells per well of a 96-well plate were cultured in DMEM overnight. The next morning the medium was replaced for DMEM containing the different metformin treatments according to figure legends and incubated for 48 hours. Cell viability was assayed using the Cell Counting Kit-8 (CCK-8)  according to the manufacturer’s protocol. Metformin (Sigma) was diluted to 1 M stock, aliquoted and frozen until its use. Assays were performed by duplicate in at least 3 independent experiments.3 × 103 cells per well of a 96-well plate were cultured in DMEM overnight. The next morning the medium was replaced for a solution  containing 5 mM 2-deoxy-D-glucose or 10 mM glucose25. After 6 hours of incubation the percentage of viable cells in 2-d-G relative to glucose was evaluated using the Cell Counting Kit-8 (CCK-8)  according to the manufacturer’s protocol. Assays were performed by duplicate in at least 3 independent experiments.6 × 1050.000 cells per well were seeded on 6-well plates. The next day medium was replaced for DMEM containing the different metformin or 2-d-G treatments. After 72 h incubation cells were trypsinized and 1,000 single viable cells were plated in 100-mm Petri dishes. The cells were then grown for 14 days and the number of colonies counted using 2% crystal violet. Assays were performed in at least 3 independent experiments.6 intact cells was measured at 37 °C using a Clark-type oxygen electrode (Hansatech Instruments) as described26. The mean of at least three independent experiments is shown.The basal respiration of 4 × 102DCFDA, Invitrogen). The mitochondrial inner membrane potential (MIMP) was evaluated using tetramethylrhodamine ester . Glucose consumption was evaluated using 2-NBDG (Molecular Probes). 2-NBDG is a derivative of glucose modified with a fluorescent amino group at the C-2 position. The C-2 position is the same position that the 18F atom is bound to in FDG. The 2-NBDG is uptaken into the cells and then decomposed into non-fluorescent forms27.Cytoplasmic ROS were assessed using 2′,7′-dichlorodihydrofluorescein diacetate  and incubation at 37 °C for 30 min in the dark, cells were collected in DMEM and analyzed immediately with a MACSQuant® (Miltenyi-Biotec) flow cytometer. Forward and side scatter were used to gate the viable population of cells, and the mean fluorescence intensity was determined with FLOWJO software (TreeStar). Assays were performed by duplicate in at least 3 independent experiments.For these assays, 0.75 × 10All the statistics were carried out using the SPSS software version 15.0. The relationship between the cumulative probability of OS, PFS and analyzed predictors, was calculated with the Kaplan-Meier method. The population was divided based on the median or tercile. The medium and high terciles for GAPDH/MTCO1 ratio were grouped since they behaved in a similar way.Bivariate correlations studies between PGC-1alpha, GAPDH/MT-CO1, and mitosis number in patient studies were analyzed using the Pearson correlation test. Differences in SUVmax levels between GAPDH/MT-CO1 ratio groups were evaluated using ANOVA.ANOVA followed by Bonferroni post-test for multiple comparisons were used to analyze the differences in clonogenicity, migration, galactose to glucose growth ratios, oxygen consumption, ROS levels, glucose consumption and PGC-1alpha or GAPDH/MT-CO1 ratio mRNA levels among the cell lines. 2-tailed Student’s t test was used to analyze differences in MIMP, cell viability and clonogenicity after metformin treatment. In all cases, values of P < 0.05 were considered statistically significant.The datasets generated during the current study are available from the corresponding author on reasonable request.


Penicillium has become one of the most attractive fungal genera for the production of bioactive molecules. Marine-derived Penicillium has provided numerous excellent pharmaceutical leads over the past decades. In this review, we focused on the cytotoxic metabolites * ; significant (IC50/LD50: 1~10 μM or 0.5~5 μg/mL); moderate (IC50/LD50: 10~30 μM or 5~15 μg/mL); mild (IC50/LD50: 30~50 μM or 15~25 μg/mL); weak (IC50/LD50: 50~100 μM or 25~50 μg/mL). The comparative potencies of positive controls were referred when they were available). produced by marine-derived Penicillium species, and on their cytotoxicity mechanisms, biosyntheses, and chemical syntheses.Since the discovery of penicillin,  Acremonium chrysogenum, thousands of bioactive metabolites have been discovered and evaluated , wh, whPenicor leads . Dimericctively) . Compounreviewed .25–30)  exhibited significant cytotoxic effects against most of the cell lines tested with IC50 values in the range of 0.89–9 μM . In contrast, brocazines C and D (27 and 28), which lack the α, β unsaturated ketone group, had much lower cytotoxicity (IC50 > 20 μM), which suggests that the conjugated ketone system is crucial to the cytotoxic properties of bisthiodiketopiperazine analogues.Four new cytotoxic bisthiodiketopiperazines (brocazines A–F) (25–30) , which sa marina . Their c31) and N-methylpretrichodermamide B (32)  (P. adametzioides AS-53) , we, we64) [68–70) . These findings indicate that terretriones might be potential anti-metastatic breast cancer candidates.In addition, three 1,4-diazepane derivatives, terretriones A, C, and D (68–70) , obtainevinaceum  and mari. CYE-87 , moderat71–76) (P. citrinum. Penicillenol B2 (74) exhibited the strongest cytotoxic activity against A-375 human malignant melanoma cell line (IC50 = 0.97 μg/mL), whereas the IC50 values of compounds 71–73 were 3.2, 13.8, and 2.8 μg/mL, respectively [75–76) showed moderate cytotoxicity against A549 cells with IC50 values of 17.2 and 12.1 μg/mL, respectively. However, penicillenols A1 and B1  showed significant cytotoxicity in HL-60 cells  [77), which contains a citrinin-like group, was isolated [77) not only induced A-375 cell apoptosis by upregulating Bax and downregulating Bcl-2, but also inhibited A-375 cell metastatic activity by suppressing matrix metalloproteinase 9 (MMP-9) and promoting the expression of its specific inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1). These findings suggest that penicitrinine A (77) is a potential lead compound.Six tetramic acid derivatives, penicillenols A1, A2, B1, B2, D1, and D2 (71–76) , were isectively ,48. Peni78–82) and quinazolinone alkaloids (83–85)  exhibited IR% values of 50–60% at 10 μg/mL. Interestingly, compound 80, which has an additional prenyl chain, showed significant cytotoxicity against MDA-MB-231 and HT-29 (human colon carcinoma) cell lines with IR% values of 92–96% at 10 μg/mL [82) exhibited significant cytotoxicity against the human lung cancer cell line 95-D (IC50 = 0.57 μg/mL). Both compounds 81 and 82 exhibited similar cytotoxicity  against HepG2 cells [83–85) showed only moderate cytotoxicity  [Quinolinone and quinazolinone alkaloids have unique pharmacophores that allow their binding to multiple sites with high affinity; moreover, they possess various biological properties . Some cy (83–85)  were isoe SD-118 . 2-quino10 μg/mL . In addiG2 cells ,54. Howel lines) ,56.P. paneum SD-44 (a deep sea sediment-derived fungus) using culture variations, three amidine anthranilic acid analogues (86–88) and one triazole anthranilic acid derivative, penipanoid A (89) . In addition, compound 88 was cytotoxic to Hela cells (IC50 = 6.6 μM) [89 with a triazole group only weakly inhibited SMMC-7721 cell viability (human hepatocarcinoma) (IC50 = 54.2 μM) while fluorouracil was used as a positive control for three cell lines with IC50 values of 25.0, 14.5, and 13.0 μM, respectively [In an ongoing study that aims to produce new active metabolites from d A (89) , were ob 6.6 μM) , whereasectively .90) , while epirubicin was used as a positive control with an IC50 value of 0.325 μg/mL [91), was first isolated from a Mediterranean sponge-derived fungus, P. chrysogenum. Sorbicillactone A (91) exhibited a selective antileukemic activity in L5178Y cells (murine leukemic lymphoblast) with an IC50 of 2.2 μg/mL, as well as in other tumor cell lines (IC50 > 10 μg/mL). The biosynthesis of sorbicillactone A (91) was investigated using 13C-labeled precursor feeding experiments, which showed that the its skeleton was derived from acetate, alanine, and methionine [92), isolated from the mangrove endophytic fungus P. brocae, showed mild lethality against brine shrimps with an LD50 of 36.7 μM, while colchicine was used as a positive control with an LD50 value of 87.6 μM [P. varibile with the DNA methyltransferase inhibitor 5-azacytidine to identify novel responsive molecules by gene silencing. A highly modified fatty acid amide, varitatin A (93), exhibited significant cytotoxicity against HCT-116 cells  and potently inhibited protein tyrosine kinases, platelet-derived growth factor receptor-beta (PDGFR-β), and ErbB4 with IR% values of 50 and 40%, respectively, at a concentration of 1 μM [94), was isolated from an endophytic fungus, P. oxalicum EN-201, derived from the marine mangrove Rhizophora stylosa. Compound 94 showed significant lethality in brine shrimps  [95), which is a known antiviral and antibiotic agent [P. notatum in 1950 [95 was isolated from the deep sea sediment-derived fungus P. commune SD-118, and showed moderate cytotoxicity in six cancer cell lines  with IC50 values of 12, 7, 10, 10, 8, and 8 μg/mL, respectively, while fluorouracil was used as a positive control with IC50 values of 4, 14, 1, 14, 0.4, and 8 μg/mL, respectively [95 induced apoptosis and autophagy by inhibiting the MEK/EPK signaling pathway and activating the class III PI3K/Beclin 1 signaling pathway [An azaphilone analogue, bis-sclerotioramin (90) , obtaine25 μg/mL . Anotherthionine . Furtherthionine . In addi 87.6 μM . Li et a of 1 μM . In addi 7.8 μM) . A previ 7.8 μM) . The isoic agent , was fir in 1950 . Recentl pathway .Penicillium is a well-known producer of eremophilane-type sesquiterpenes with phytotoxic, mycotoxic, and phytohormonic activities [Penicillium sp. PR19 N-1, yielded three new cytotoxic eremophilane-type sesquiterpenes (96–98)  and A549  cancer cell lines [99–101) were isolated from a sea mud-derived fungus, Penicillium sp. BL 27-2. Of these, compound 99 was the most cytotoxic to P388, A549, HL-60, and BEL-7402 cell lines , whereas compounds 100 and 101 had IC50 values in the range of 3–12 μM [102), isolated from a marine sponge-derived fungus, P. adametzioides AS-53, displayed selective cytotoxicity against NCI-H446 cell lines (IC50 = 5 μM), compared to its cytotoxicity against the other 13 tumor cell lines tested , L02 (human hepatocarcinoma), LM3 (murine breast cancer), MA (mouse Leydig tumor), MCF-7, SGC-7901, SW1990, SW480, and U251) (IC50 > 10 μM) [The genus tivities ,69. Chem (96–98) , which wll lines ,71. Thre 3–12 μM . These r> 10 μM) .Penicillium sp. was reported to be a good source of cytotoxic diterpenes. Six tetracyclic diterpenes, conidiogenones B–G (103–108) (104) was potently cytotoxic against HL-60 and BEL-7402 cells with IC50 values of 0.038 and 0.97 μM, respectively; however, it was not cytotoxic against A549 and MOLT-4 cell lines at 50 μM. Other conidiogenones  had moderate cytotoxicity with IC50 values ranging from 1 to 50 μM [109–110) were also obtained from this fungus and showed cytotoxicity comparable to that of cisplatin (the positive control) against MCF-7 cells  [The deep sea sediment-derived fungus 103–108) , exhibitto 50 μM . The spisplatin) .111) (P. chrysogenum QEN-24S. Penicisteroide A (111) displayed moderate cytotoxicity against Hela, SW1990, and NCI-H460 cell lines with IC50 values of 15, 31, and 40 μg/mL, respectively [112–114) and two epidioxygenated steroids (115–116) were isolated from the marine moss-derived fungus Penicillium sp. These steroids moderately inhibited HepG2 cell line growth  [117), produced by a sea squirt-derived fungus, P. stoloniferum QY2-10, was cytotoxic to P388 cells with an IC50 of 4.07 μM [Penicillium sp. fungus collected from the inner tissues of an unidentified sponge is reportedly the source of two epimeric steroids (118–119) and two cytotoxic steroids of a new class, dankasterone A (120) and B (121). Dankasterone A (120) was more effective than the positive control, adriamycin (IC50 = 0.98 μM) against HL-60, Hela, and K562 cancer cell lines with IC50 values of 0.78, 4.11, and 7.57 μM, respectively. Compounds 118–119 and 121 also significantly inhibited K562 cell growth  [Although several marine-derived steroids have been isolated, few have been found to be bioactive. A cytotoxic polyoxygenated steroid, penicisteroide A (111) , was isoectively . Three octively) . In addi 4.07 μM . Moreovectively) .122–124) , SKMEL-2 (human skin cancer), XF498 (human CNS cancer), and HCT15 (human colon cancer)) with IC50 values in the range of 3–10 μg/mL, whereas compound 124 had IC50 values ranging from 20 to 40 μg/mL . These results suggest that the quinone form tends to be less cytotoxic [125), isolated from marine-derived Penicillium sp. F11., contains a carboxylic acid group instead of the isoprenyl tail, which resulted in mild cytotoxicity against fibrosarcoma (HT1080) and human nasopharyngeal carcinoma (Cne2) cell lines  [Meroterpenes are widely distributed in the marine environment, particularly in brown algae and microorganisms. Terpene-quinone and -hydroquinone are the major bioactive members because they produce reactive oxygen species (ROS) . Three q122–124)  were isoytotoxic . Penicilcoma HT10 and huma126–127) (P. concentricum ZLQ-69 and displayed moderate and selective cytotoxicity against MGC-803 cells (human gastric cancer) with IC50 values of 19.1 and 13.6 μM, respectively  [Penicillium sp. F446 yielded two new sesquiterpene γ-pyrone-type meroterpenes, penicillipyrone A and B (128–129), which were moderately cytotoxic against A549 cells  [130–131), isolated from the marine mangrove endophytic fungus Penicillium 303#, exhibited IC50 values of 20–30 μg/mL in four cancer cell lines , while epirubicin was used as a positive control with IC50 values of 0.2~0.6 μg/mL [Aspergillus fumigatus in 1949, and has been used as an antimicrobial [132), a natural chlorinated merosesquiterpene related to fumagillin, was obtained from a marine-derived Penicillium sp., and showed selective in vitro antiproliferative activity against osteosarcoma cell lines , while doxorubicin was used as a positive control with IC50 values of 0.04~2 μM [132) exhibited stronger cytotoxicity against human osteosarcoma SaOS2 and MG63 cancer cell lines. However, its cytotoxicity was less than that of TNP470  [Two sesquiterpene α-pyrones, phenylpyropenes E and F (126–127) , were is0.37 μM) . Further 1.2 μM) . Two pol.6 μg/mL . Fumagilicrobial . Recentl.04~2 μM . Ligerinnalogue) .133–134) and A, B (135–136) , these four natural chromones (133–136) displayed significant cytotoxicity against eight carcinoma cell lines (human lung adenocarcinoma (H1975), human lymphoma (U937), K562, BGC823, MOLT-4. MCF-7, HL-60, and Huh-7) (IC50 < 10 μM). Of these, oxalicumone A (135) was the most cytotoxic against MOLT-4 cell line (IC50 = 0.30 μM). An SAR study showed that the 2,3-dihydrothiophene unit was crucial for activity and that the presence of 1-OH and absolute configuration at C-6 contributed to cytotoxicity [135) inhibited leukemia cell growth and induced apoptosis, in part, via the induction of the endoplasmic reticulum stress pathway by upregulating calnexin and Bax and activating unfolded protein response [135) could induce oxidative stress injury in the mitochondria, and thus promote human renal epithelial cell death [145), a novel cyclopentachromone sulfide which is structurally similar to dihydrothiophene-condensed chromones, was isolated from a neomycin-resistant mutant of the marine-derived fungus P. purpurogenum G59, and showed selective cytotoxicity against HL-60 cancer cell line (IC50 = 16.7 μM) [146), a chiral dimeric tetrahydroxanthone, was first isolated from Aspergillus sp. before discovering that the deep sea sediment-derived fungus Penicillium sp. F11 is a good source of this compound. Compound 146 induced HL-60 cell apoptosis by modulating the Rho GDP dissociation inhibitor 2 pathway [146) could induce apoptosis by activating caspase 3 and 9 through the mitochondrial pathway in hepatocellular carcinoma, wherein it was found to be more effective than 5-fluorouracil [147), obtained from a gorgonian-derived fungus, P. chrysogenum, exhibited selective cytotoxicity against Hela and rhabdomyosarcoma cell lines  while adriamycin was used as a positive control with IC50 values of 0.43 and 0.09 μM, respectively [Chromone derivatives are abundantly present in nature and are considered potential immunomodulatory, anticancer, and anti-inflammatory agents. Chromone scaffolds were reported to possess outstanding pharmacological properties . A Chine135–136)  from a mtoxicity ,88. Subsresponse . Anotherll death . Chromos16.7 μM) . Secalon pathway . Recent ymphoma U7, K562, ectively .148–150) (P. chrysogenum SCSIO 41001), a marine sponge-derived fungus Penicillium sp., and a mangrove endophytic fungus (Penicillium sp. ZH16), respectively. The dimeric isocoumarin, bipenicillisorin (148), displayed significant cytotoxicity against K562, A549, and Huh-7 cell lines , while taxol was used as a positive control with IC50 values of 3.44, 2.61, and 14.70 nM, respectively [149) exhibited significant cytotoxicity against L5178Y cells (a murine lymphoma cell line) with an IC50 value of 8.4 μM  [150) showed moderate cytotoxicity against human nasopharyngeal carcinoma (KB and KBv200) cell lines  [Coumarin derivatives of the chromone isomers (148–150)  were alsectively . The dih 4.3 μM) . Moreovectively) .151) (P. citrinum in 1931 [151 is strongly nephrotoxic because of its inhibition of respiration complex III [151 was further investigated [Penicillium sp. FF001 was found to be a good source of unique and potent citrinin derivatives [152–153), isolated from the marine sediment-derived fungus P. citrinum, showed moderate cytotoxicity against a human Caucasian colon adenocarcinoma cell line (SW-620)  [154), with a novel spiroketal structure, isolated from an acid mine lake fungal extremophile Penicillium sp., showed selective and extraordinary cytotoxicity against a human ovarian carcinoma cell line (OVCAR-3) at nanomolar concentrations (GI50 = 91 nm) [154) was previously described [P. thomii, yielded a new citrinin analogue, sargassopenilline C (155), which possessed a unique 6,6-spiroketal skeleton and inhibited the transcription of oncogenic nuclear factor, AP-1 (IC50 = 15 μM) [156–157), isolated from a marine sponge-derived fungus Penicillium sp., inhibited both DNA polymerases (α and β) [158), a marine sediment-derived fungal metabolite (P. citrinum) containing a rare carbon-bridge citrinin dimer, induced A-375 cell apoptosis by generating ROS via a caspase-related pathway [159–160) possibly biogenetically derived from citrinin were found. Perinadine A (159), a scalusamide A-type pyrrolidine isolated from a fish gastrointestinal fungus, P. citrinum, exhibited mild cytotoxicity against a murine leukemia L1210 cell line (IC50 = 20 μg/mL) [160), obtained from a marine sediment-derived fungus, Penicillium sp. F011, possessed a novel pyrrole-containing phenalenone moiety and demonstrated weak cytotoxicity against A549 cells (IC50 = 67.3 μM), while doxorubicin was used as a positive control with an IC50 value of 3.3 μM [Citrinin (151) , a typic in 1931 . Compounplex III . The biostigated . Interesivatives . Two newctively) . One pen= 91 nm) . The totescribed . An alga= 15 μM) . Two pheα and β) . Dicitri pathway . In anot0 μg/mL) . Howeverf 3.3 μM .161–163) (164–166), which were isolated from a marine sediment-derived fungus, P. commune QSD-17 (comazaphilones D–F), and a marine seaweed-derived P. pinophilum Hedgcok (pinophilins A-B and Sch 725680). Comazaphilones D–F (161–163) showed selective but weak cytotoxicity against SW1990 cell line , while fluoruoracil was used as a positive control with an IC50 value of 120 μM) [164–166) were suggested to suppress cancer cell proliferation by inhibiting DNA replication via the inhibition of mammalian DNA polymerases A, B, and Y [Other fungal azaphilone polyketides include comazaphilones D–F (161–163) , pinophi 120 μM) . AzaphilB, and Y .Penicillium sp. strain OUPS-79, which is derived from the marine alga Enteromorpha intestinalis, yielded various cytotoxic polyketides, including penostatins A–C, E–I  . However, penostatin D (170) exhibited moderate cytotoxicity (ED50 = 11.0 μg/mL), which may be attributed to the absence of the cyclic conjugated enone system. Moreover, penostatin C (169) exhibited significant cytotoxicity in seven of the 36 cell lines tested with ED50 values ranging from 1 to 2 μg/mL. Recent studies have shown that penostatins A–C (167–169) may be tyrosine phosphatase 1B (PTP1B) inhibitors, which can be used to treat type II diabetes and other associated metabolic diseases , while sodium orthovanadate was used as a positive control with an IC50 value of 0.65 μM [167, 168, 172) was previously reported [171–175)  [111,112 0.65 μM . The totreported ,115.176)  was obtained from the co-cultivation of marine mangrove-derived Penicillium sp. WC-29-5 and Streptomyces fradiae 007. Compounds 177–178 displayed significant cytotoxicity against H1975 cell lines . Moreover, compound 178 exhibited cytotoxicity against HL-60 cells (IC50 = 3.73 μM) [179) as well as griseophenones I and G (180–181), were isolated from a marine-derived P. canescens. They inhibited the growth of KB cells at a concentration of 0.6 μM  [182), and two diphenyl ether derivatives, penikellides A and B (183–184), were isolated from a mangrove endogenous fungus, Penicillium sp. MA-37. These three metabolites exhibited moderate brine shrimp lethality , while colchicine was used as a positive control with an LD50 value of 1.22 μM [185), a multifunctional metabolite produced by a marine sediment-derived Penicillium sp. strain, was shown to be an acyl-CoA cholesterol acyltransferase (ACAT) [185 was found to exhibit cytotoxic, antibiotic, and plant growth inhibitory properties. Recently, two marine fungi, P. pinophilum (derived from a gorgonian) and Penicillium sp. ZLN29 (derived from a sediment), were found to produce penicillide (185) and penicillide derivatives (186–187) that exhibited potent cytotoxicity against HepG2 cell line ; moreover, compound 187 showed additional cytotoxicity against Hela cell line (IC50 = 6.1 μM) [188) and averantin (189), isolated from a marine sediment-derived fungus, P. flavidorsum SHK1-27, were cytotoxic against K562 cell line , while adriamycin was used as a positive control with an IC50 value of 1.5 μM. Nidurufin (188) was suggested to induce cell cycle arrest at the G2/M transition in a time-dependent manner [188), an aflatoxin precursor, was previously described [Fungal phenolic polyketides have diverse biological activities and unique structures . A weak 176) , was obtum HSY05 , whereas3.73 μM) . Using actively) . Further 1.22 μM . Penicile (ACAT) , nonpepte (ACAT) , and oxye (ACAT) . Further 6.1 μM) ,125. Twot manner . The totescribed .190–191)), two bisvertinolones (192–193), and three bridged bicyclic bisorbicillinoids (194–196) (P. terrestre. Dihydrobisvertinolone (192) and trichodimerol (196) demonstrated the strongest cytotoxic effects , while etoposide was used as a positive control with IC50 values of 1.4 and 0.064 μM, respectively [197–198). Interestingly, the configuration at C-19 was found to largely determine the cytotoxicity, wherein chloctanspirone A (197) (R configuration) was 4-fold more active than chloctanspirone B (198) (S configuration) in HL-60 and A549 cancer cell lines [Members of the sorbicillinoid family are hexaketide metabolites isolated from various fungi. In 2005, Zhu et al. found two sorbicillin analogues (benzoquinone (194–196)  in a marectively ,129. Theectively . Furtherll lines .200) -10,11-Dehydrocurvularin (199) was isolated from marine sponge-derived Penicillium sp. DRF2 and Curvularia sp. It exhibited significant cytotoxicity with mean IC50 values ranging from 0.28 to 6 μM in 14 different solid tumors (36 tumor cell lines) [Penicillium fungi are also a good source of tanzawaic acid polyketides, which exhibit antibiotic resistance [201), isolated from a marine-derived fungus, Penicillium sp. CF07370, was selectively toxic to U937 cancer cells via the activation of the mitochondrial apoptotic pathway [202), isolated from P. steckii, effectively and selectively bound to the transcription factor, forkhead box O1 (FOXO1), which can regulate epidermal growth factor receptor (EFGR) signaling, suppress cell cycle progression, and stabilize the conformation of FOXO1-DNA [Macrolides represent a well-known class of antibiotics, and curvularin (200)  is a heal lines) ,134. Pensistance , as wellsistance  and cyto pathway . ComputaOXO1-DNA .210–211) , L1210 , and KB cells . Recently, seven new similar lipopeptides, penicimutalides A–G (203–209) and fellutamides B and C (211–212) were isolated from a diethyl sulfate-induced mutant of the marine fungus, P. purpurogenum G59 [203–209 and 212 exhibited weak cytotoxicity . However, fellutamide B (211) with a C-terminal aldehyde group was more potent with IC50 values that ranged from 20 to 80 μg/mL, which indicated that the C-terminal aldehyde group improves the cytotoxicity.Fellutamides A and B (210–211)  were thellutanum . Compounenum G59 . They wePenicillium sp. is a good source of polyphenol derivatives. Two trimeric peniphenylanes A, B (213–214) and three dimeric peniphenylanes D, F, G (215–217) (P. fellutanum HDN14-323. Peniphenylane D (215) displayed more potent and extensive cytotoxicity with IC50 values in the range of 9–30 μM in three cancer cell lines , while doxorubicin was used as a positive control with the IC50 values of 0.2, 0.6, and 0.2 μM, respectively [P. terrestre was found to produce several gentisyl alcohol derivatives, including trimeric terrestrol A (225) and dimeric terrestrols B–H (218–224), which were found to be cytotoxic against HL-60, MOLT-4, BEL-7402, and A549 cancer cell lines with IC50 values in the range of 5–65 μM [P. expansum 091006 yielded four novel cytotoxic phenolic bisabolane sesquiterpenoids ) with IC50 values of 15.7, 5.4, 18.2, and 20.8 μM, respectively, in HL-60 cells. In addition, expansol B (227) showed significant cytotoxicity against A549 cells (IC50 = 1.9 μM), while etoposide was used as a positive control with IC50 values of 0.042 and 0.63 μM for two cell lines, respectively [Polyphenol derivatives are the most abundant fungal secondary metabolites. Unsurprisingly, marine 215–217)  were isoectively . The mar 5–65 μM . Interesectively ,144.230) (Penicillium sp. was found to produce patulin (230) along with (+)-epiepoxydon (231), both of which exhibited extraordinary cytotoxic effects in P388 cells . Furthermore, (+)-epiepoxydon (231) had significant cytotoxicity against seven other cancer cell lines with IC50 values in the range of 0.3–1.5 μg/mL [232) isolated from a mangrove endophytic Penicillium sp. displayed moderate cytotoxicity against KB and KBV200 cells  [233), isolated from marine-derived Penicillium strain, exhibited moderate cytotoxicity against POS1, AT6-1(murine prostatic carcinoma), and L929 (murine fibroblasts) cell lines  while doxorubicin was used as a positive control with IC50 values of 0.04~2 μM [Patulin (230)  is a myc.5 μg/mL . The isoctively) , whereas.04~2 μM .154 in this review inhibited OVCAR-3 cell line at nanomolar concentrations). Furthermore, the activation of silent gene clusters may activate new biosynthetic pathways that produce compounds with novel structure, which provide equally valid leads . Interestingly, the halogenation of compound 31, which was completely inactive, produces compound 32, which exhibited a much greater potency (compound 32 had significant cytotoxicity in 22Rv1 cells at nanomolar levels) [The rapid development of marine biotechnology and ever increasing needs of industrial applications resulted in the emergence of marine natural products as alternative drug sources in the early 1990s . Marine- levels) .Penicillium has been explored for antitumor leads in recent years [Penicillium fungus and chemically synthesized analogues. Of these, the major metabolites are alkaloids, particularly diketopiperazine alkaloids and indole alkaloids (50 < 1 μg/mL). Furthermore, a series of diketopiperazine alkaloids, gliotoxin analogues, and roquefortine analogues with remarkable cytotoxicity at nanomolar levels are potential anticancer leads. Terpenoid metabolites appear to be more effective against cancer cell lines than steroids; in particular, compounds 99, 104, and 132 were effective at nanomolar levels. Furthermore, citrinins  and their derivatives, which are polyketide mycotoxins, possess excellent cytotoxic activities. Penostatins (cytotoxic polyketides) are cytotoxic to P388 cells with IC50 values of ~1 μg/mL. With the exception of 210 and 211, lipopeptides exhibited moderate cytotoxicity. In addition, the Penicillium genus can produce polyphenolic compounds (terrestrols) with pronounced cytotoxicity.The genus nt years . Howeverlkaloids . CytochaPenicillium sp. Different marine hosts and environments can also affect the biosynthesis of metabolites by endozoic fungi. Notably, over 99% of the symbiotic microorganisms cannot be cultured. Further investigations may utilize metagenome libraries of the host organisms to identify more metabolites produced by symbiotic microorganisms [Although our review includes most of the cytotoxic metabolites described in the literature, more compounds are yet to be identified in marine rganisms . Additio


Human cancer cell lines are used in research to study the biology of cancer and to test cancer treatments. Recently there are already some large panels of several hundred human cancer cell lines which are characterized with genomic and pharmacological data. The ability to predict drug responses using these pharmacogenomics data can facilitate the development of precision cancer medicines. Although several methods have been developed to address the drug response prediction, there are many challenges in obtaining accurate prediction.Based on the fact that similar cell lines and similar drugs exhibit similar drug responses, we adopted a similarity-regularized matrix factorization (SRMF) method to predict anticancer drug responses of cell lines using chemical structures of drugs and baseline gene expression levels in cell lines. Specifically, chemical structural similarity of drugs and gene expression profile similarity of cell lines were considered as regularization terms, which were incorporated to the drug response matrix factorization model.We first demonstrated the effectiveness of SRMF using a set of simulation data and compared it with two typical similarity-based methods. Furthermore, we applied it to the Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Cell Line Encyclopedia (CCLE) datasets, and performance of SRMF exceeds three state-of-the-art methods. We also applied SRMF to estimate the missing drug response values in the GDSC dataset. Even though SRMF does not specifically model mutation information, it could correctly predict drug-cancer gene associations that are consistent with existing data, and identify novel drug-cancer gene associations that are not found in existing data as well. SRMF can also aid in drug repositioning. The newly predicted drug responses of GDSC dataset suggest that mTOR inhibitor rapamycin was sensitive to non-small cell lung cancer (NSCLC), and expression of AK1RC3 and HINT1 may be adjunct markers of cell line sensitivity to rapamycin.Our analysis showed that the proposed data integration method is able to improve the accuracy of prediction of anticancer drug responses in cell lines, and can identify consistent and novel drug-cancer gene associations compared to existing data as well as aid in drug repositioning.The online version of this article (doi:10.1186/s12885-017-3500-5) contains supplementary material, which is available to authorized users. Patients suffering from the same cancer may differ in their responses to a specific medical treatment. Precision cancer medicines aim to decipher the cause of a given patient’s cancer at the molecular level and then tailor treatment to address that patient’s cancer progression . IdentifMachine learning algorithms such as elastic net regularization and random forests were used to search for genomic biomarkers of drug sensitivity in cancer cell lines for individual drugs –5, 9, 10Based on the fact that similar cell lines and similar drugs exhibit similar drug responses , here weWe firstly used the data from the Genomics of Drug Sensitivity in Cancer project consisting of 139 drugs and a panel of 790 cancer cell lines (release-5.0). Experimentally determined drug response measurements were determined by log-transformed IC50 values . Notably, a lower value of IC50 indicates a better sensitivity of a cell line to a given drug. In addition, cell lines were characterized by a set of genomic features. We selected the 652 cell lines for which both drug response data and gene expression were available. Furthermore, we focused on the 135 drugs for which SDF format  were available from the NCBI PubChem Repository. Then PubChem fingerprint descriptors were computed using the PaDEL software . The resThe data from the Cancer Cell Line Encyclopedia consists of 24 drugs and a panel of 1036 human cancer cell lines. Drug sensitivity data were summarized by activity area (the area over the drug response curve). Notably, the higher the activity area value, the better the sensitivity. We selected the 491 cancer cell lines for which both drug sensitivity measures and gene expression profile data were available. There are 23 drugs having PubMed SDF files from which we can obtain drug chemical structures. The resulting drug response matrix of 23 drugs by 491 cell lines has 11,293 entries, out of which 423 (3.75%) are missing and 10,870 are known.m drugs and n cell lines into a shared latent space, with a low dimensionality K, where K ≪ min . The properties of a drug di and a cell line cj are described by two latent coordinates ui and vj, respectively. As to drug response matrix Y, we aimed to approximate each known response value of drugdi for cell line cj via their latent coordinates which can be our objective function:W is a weight matrix, in which Wij = 1 if Yij is a known response value; otherwise Wij = 0, W ⋅ Z denotes the Hadamard product of two matrices W and Z, U and V are two matrices containing ui and vj as row vectors, respectively, and ∣|⋅|F is the Frobenius norm.In this article, we applied a powerful matrix factorization framework to predict anticancer drug responses in cell lines Fig. . SimilarU and V to training data, L2 (Tikhonov) regularization was imposed to the latent variables U and V.Then to avoid overfitting of Sd and Sc are drug similarity matrix and cell line similarity matrix, respectively.Furthermore, prior knowledge on drugs and cell lines is very useful and valuable to decipher the global structure of drug-cell line response data. Based on the results that similar cell lines and similar drugs exhibit similar drug responses , we propThe final drug response prediction model can be formulated by considering the drug response matrix as well as the similarity of drugs and cell lines. By plugging Eqs  and 4)  4) into U and V, we searched for the local minimum instead of the global minimum by an alternating minimization algorithm. The algorithm which was deduced detailedly in Additional file U and V alternately. We provided this algorithm in the following, and the software can be freely downloaded from the website (https://github.com/linwang1982/SRMF).Since the objective function (5) is not convex with respect to variables n is the number of cell lines with known response values for drug D, R and D versus cell line C, respectively. Moreover, drug-averaged PCC and RMSE are computed as the average PCC and RMSE over all drugs.By accounting for variability in sensitive ranges of drugs, the correlation between observed and predicted response values for all drug response entries may overestimate the prediction performance . Here, wThere is compelling evidence that the sensitive and resistant cell lines of each individual drug are more valuable to decipher mechanisms of drug actions, we also care about PCC and RMSE from sensitive and resistant cell lines for each drug, and they were denoted as PCC_S/R and RMSE_S/R, respectively. Here, for each drug the logIC50 (activity area) were split into quartiles, with cell lines in the first and fourth representing drug-sensitive (−resistant) and –resistant (−sensitive) cell lines, respectively, which was also performed for drug sensitive analysis of breast cancer cell lines . ConsequK was set as 45 for GDSC dataset  at 0.001 increments and 0.01 increments, respectively. The weight parameter λ was selected from range of  at 0.01 increments [The settings of the hyper-parameters of each method were as follows. For the matrix factorization based methods, including SRMF and KBMF, the low dimensionality  dataset . Moreovecrements . For a pcrements .We calculated the Pearson correlation between each pair of gene expression profiles of cell lines after normalizing gene expression values across cell lines. As shown in Fig. A hierarchical clustering of 135 drugs based on their chemical fingerprint features was performed   inhibitor lapatinib, where more than half of response values (342/652) were missing, and a cyclin D kinases (CDKs) 4 and 6 inhibitor PD-0332991, where nearly 10% of response values (62/652) were missing. There were clear associations between EGFR and ERBB2 mutations and sensitivity to lapatinib that targets the product of these genes , 25. HerThe newly predicted drug responses combined with existing drug responses were able to detect novel drug-cancer gene associations as well. For example, MET amplification was significantly associated with sensitivity to c-Met inhibitor PHA-665752 , 28, whiP value=1.33 × 10‐10). Similar situation appeared with HINT expression . Interestingly, AK1RC3 has been suggested as an adjunct marker for differentiating small cell carcinoma from NSCLC [The newly predicted drug responses of GDSC dataset can aid in drug repositioning. The mTOR inhibitor rapamycin was sensitive to non-small cell lung cancer (NSCLC)  based onom NSCLC , and theom NSCLC .Fig. 8ReSRMF currently incorporated the gene expression profile based cell line similarity. Notably, SRMF can be extended to incorporate multiple types of similarity measures for cell lines through weighted low-rank approximation  and multIn this study, we developed a similarity-regularized matrix factorization method SRMF to predict the response of cancer cell lines to drug treatments for IC50 values in the GDSC and activity areas in the CCLE study. The performance of SRMF was first evaluated through simulation studies and further validated by the 10-fold cross validation on GDSC and CCLE datasets. Clearly, SRMF shows better overall prediction performance than other methods in the comparison study. Finally, in comparison with existing data, the newly predicted drug responses of GDSC dataset can find consistent and novel drug-cancer gene associations and aid in drug repositioning.Additional file 1:U and V by alternating minimization algorithm. The derivation process of the updating formulas is described in detail. (PDF 192 kb)Obtaining the updating formulas of Additional file 2:The hierarchical clustering of drugs in GDSC dataset based on their PubChem fingerprint descriptors. The similarity between pair fingerprint descriptors of drugs was measured by the Jaccard coefficient. The scale to the left of the dendrogram depicts the distance value (1-Jaccard coefficient) represented by the length of the dendrogram branches connecting pairs of node. The distance threshold was specified to 0.29 to group the drugs into clusters. (PDF 9 kb)Additional file 3:A set of simulated data used to evaluate the prediction performance of SRMF. Target drug responses, their perturbations with similarities of drugs and cell lines used as inputs for SRMF are simulated. Besides, an example for illustrating the efficiency of SRMF is described in detail. (PDF 185 kb)Additional file 4:Prediction performance comparisons of four methods for the drugs targeting genes in the ERK pathway with respect to two measurements. A) Pearson correlation coefficient between predicted and observed response values of sensitive and resistant cell lines for each drug. B) Root mean squared error between predicted and observed drug responses of sensitive and resistant cell lines for each drug. (PDF 381 kb)


The effects of this agent on cell cycle and apoptosis were investigated. Protein markers relevant to these mechanisms were also assessed. We found that the inhibitory effect of dsBPT on lung and head and neck cancer cell growth (IC50 ranged between 0.1–0.2 μM) was 10 times greater than that on normal epithelial cells. dsBPT alone induced autophagy, G1 cell cycle arrest, and apoptosis. Our in vivo studies indicated that dsBPT inhibited tumor growth in a dose-dependent manner in a head and neck cancer xenograft mouse model. The combination of dsBPT with cisplatin synergistically inhibited cancer cell growth with a combination index of 0.3. Moreover, the combination significantly reduced tumor volume as compared with the untreated control (p = 0.0017) in a head and neck cancer xenograft model. No organ related toxicities were observed in treated animals. Our data suggest that dsBPT is a novel and potent antitumor drug that warrants further preclinical and clinical development either as a single agent or in combination with known chemotherapy drugs such as cisplatin.The anti-tumor effect of a chelating phen-based ligand 2,9-di- Rphen) ligands have been used to synthesize gold (III) coordination complexes possessing enhanced biological stability. Several recent studies have addressed the antiproliferative potential of 1,10-phenanthroline (phen)-based ligands and their metal complexes in other types of cancer cells [in vitro [sec-butyl-1,10-phenanthroline (dsBPT). Augmenting our recent report in which this compound was tested for activity against glioblastoma tumor [Cisplatin has been widely used over the past 30 years to successfully treat a variety of cancers, including testicular, ovarian, head and neck, and bladder cancers –4. Despier cells –11. Previn vitro –15. Therma tumor , the worin vitro and in vivo antitumor studies in a variety of head and neck and lung tumor cell lines. Cell viability assays revealed that dsBPT possessed significant anti-proliferation activity against head and neck and lung cancer cell lines, including one tumor cell line that has been previously shown to be resistant to cisplatin. These results prompted the investigation of the antitumor properties of dsBPT against mouse xenograft tumors generated from two of the cancer cell lines. Additional drug effect studies and cell signaling pathway analyses were carried out in order to gain insight about the possible mechanism through which this drug induces tumor cell death. Finally, a combination treatment comprised of dsBPT and cisplatin was evaluated in both in vitro tumor cell cultures and tumors grown in mouse models. The results of the assessment of the in vivo antitumor activity of dsBPT, the in vivo antitumor activity of the dsBPT-cisplatin combination therapy, and the potential tumor cell death pathways initiated by this compound are described herein.In this study, the dsBPT chelator was evaluated in a series of dsBPT was synthesized and purified according to a previously published protocol . The pur2 humidified incubator. The HNSCC cell lines Tu212 and Tu686 were kindly provided by Dr. Gary L. Clayman  in 2002 and Dr. Peter G. Sacks  in 2013, respectively [All head and neck squamous cancer cell lines (HNSCC) were maintained in DMEM/F12 (1:1) medium supplemented with 10% heat-inactivated fetal bovine serum in a 37°C, 5% COectively . The humectively . These tectively . All celet. al.[Sulforhodamine B (SRB) cytotoxicity assays were performed as described by Skehan et. al.. Cells wTM SC flow cytometer (Beckman Coulter). For cell cycle analysis, cells were exposed to equal concentrations of dsBPT and cisplatin (2 μM for A549 and 1 μM for Tu212) and stained with PI 24h and 48h after drug treatment.Cell apoptosis and cell cycle analyses were performed by flow cytometry. A549 and Tu212 cells were exposed to 8 μM and 4 μM dsBPT, respectively. Cells were harvested at 24, 48, and 72 h, and cell apoptosis was determined by Annexin-5 staining method using a Cell Lab Quanta3VO4, 10mmol/L β-glycerophosphate and 1% Protease Inhibitor Cocktail . Protein (20–30 μg) was separated on 8–12% SDS-PAGE, transferred onto a polyvinylidene difluoride membrane  and the desired protein was probed with specific antibodies–DR5 , p21 , p27 , caspase 3 , and caspase 8 , Mouse anti-actin antibody  was used as a sample loading control. Immunostained protein bands were detected with an enhanced chemiluminescence kit .Whole cell lysates were extracted from drug-treated cells using lysis buffer containing 50mmol/L HEPES buffer, 150mmol/L NaCl, 1mmol/L EDTA (pH 8.0), 1mmol/L EGTA (pH8.0), 1% IGEPAL CA-630, 0.5% Triton X-100, 10mmol/L NaF, 2mmol/L NaTo test the cytotoxicity of dsBPT in tumor cells, xenografted tumor tissues were fixed in paraformaldehyde and subjected to paraffin embedment. Embedded samples were cut to a thickness of 8μm. IHC staining of Ki67 was performed using the R.T.U. Vectastain kit following the manufacturer’s standard protocol . Tissue sections were incubated with mouse anti-human Ki-67 antibody  overnight at 4°C. The slides were stained with 3,3 diaminobenzidine  and counterstained with hematoxylin . For TUNEL assay, frozen tumor samples were kept in OCT, and assayed using the TUNEL kit  following the manufacturer’s procedure. The slides were counterstained with DAPI .nu/nu, Taconic, NY), aged 4–6 weeks (~20 g body weight), were used to test the in vivo effects of dsBPT. Tu212 or A549 cells (2 x 106) were injected into the right flank of the mice subcutaneously. The tumor volume was calculated using the formula: V = π/6 x larger diameter x 2, as reported previously [All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Emory University. Nude mice (athymic eviously . Growth 2 gas or other methods: (1) weight > 25% (Score = 18); (2) weight loss > 20  plus decreased activity, or hunched posture (Score = 3); (3) epithelial tumor > 2.0 cm in length (Score > 12) plus decreased activity, or hunched posture (Score = 3); (4) epithelial tumor > 1.8 (Score = 12) and weight loss = 15% (Score = 12). Mice were euthanized with CO2 in a chamber that allows for their visualization, using a flow rate of 10–30% of the chamber volume per minute as controlled with a pressure-reducing regulator and a flow meter. All animals were checked for lack of breathing for at least 5 minutes prior to being placed in carcass cooler.Mice injected with tumor cells may develop illness symptoms, such as cachexia, respiratory distress, hunching over, ataxia, inability to mobilize around cage, or inability to either eat or drink. Mice were examined twice per week to determine the Tumor Burden Score established by the Emory IACUC. In any of the following cases, mice were euthanized by COTwo sets of animal experiments were performed. First, animals were treated with different doses of dsBPT, including 2 mg/kg, 5 mg/kg, and 10 mg/kg and the PBS control. The animals were continuously administered the agents 3 times a week by tail vein injection. Tumor size and animal weight were measured 3 times every week. In the second experiment, the combination effect of dsBPT and cisplatin was examined. The experimental groups were (i) PBS control, (ii) 2 mg/kg cisplatin, (iii) 2 mg/kg dsBPT, and (iv) 2 mg/kg of both cisplatin and dsBPT. Drugs were given intravenously every 6 days. Tumor size and animal weight were measured every 3 days. When the tumor size of the control group reached the IACUC endpoint, the animals were sacrificed, and the tumors were harvested for Ki67 IHC and TUNEL staining. Major organs were also harvested for H&E staining to evaluate the potential toxicity of the drug. For drug toxicity assays, mouse blood was subject to biochemical tests for alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine and blood urea nitrogen concentrations. Samples were sent to the Clinical Pathology Laboratory, College of Veterinary Medicine, University of Georgia for these tests.The mice were randomly assigned to different treatment groups (described above) and control groups. The mixed effects model was mainly used to examine the tumor growth difference between each of the treatment groups and control. The fixed-effect terms in the model were groups, time, and interaction between groups and time, which allows different tumor growth rate by groups. The within subject covariance structure was chosen based on AIC and BIC criteria . Box-Cox data transformation was applied to the outcome to ensure the fitted model met statistical assumptions. Statistical analysis was performed using SAS 9.2. The critical significance level of 0.05 was chosen.The structure of dsBPT is illustrated in To identify the mechanism of cell inhibitory effect of dsBPT, we examined possible alterations in cell cycle distribution and protein markers relevant to cell cycle regulation and cell growth arrest. It was found that treatment with dsBPT at 1–2 μM concentrations resulted in significantly higher levels of G1 cell cycle arrest than seen following cisplatin treatment in both A549 and Tu212 cancer cell lines at both 24 and 48 hour time points (p< 0.05), while cisplatin significantly induced G2 cell cycle arrest (p <0.05) at 24 and 48 hour time points .Flow cytometry was used to quantify the level of apoptosis in A549 and Tu212 cancer cells upon treatment with dsBPT. We did not observe cell apoptosis when the same concentrations (1–2 μM) as used in cell cycle analysis were applied . HoweverIn order to identify the mechanism underlying the induction of apoptosis and cell cycle arrest by dsBPT, we carried out Western blot analyses to detect alterations in signaling pathways relevant to the regulation of cell cycle progression and apoptosis in A549 and Tu212 cell lines after treatment with the drug for 24, 48 and 72 hours. Increased levels of caspase 8 were found in both A549 and Tu212 tumor cell lines when treated with dsBPT . Given t6 Tu212 cells. dsBPT was administered by tail vein injection at 0mg/kg, 2mg/kg, 5mg/kg, and 10mg/kg doses. One group of mice treated with PBS was also included as a control. Each group included 6 mice. Tumor volume and mice body weight were measured every 3 days. dsBPT significantly inhibited Tu212 xenograft tumor growth at doses of 5mg/kg and 10 mg/kg as compared with the control (both doses p<0.04)  . IHC and p<0.04) . There w p<0.04) .in vivo experiments , we speculated that combination treatment using both dsBPT and cisplatin may increase the tumor killing effect. As expected, the combination of dsBPT with cisplatin synergistically enhanced the inhibitory effect of both drugs as indicated by CIs of less than 1.0 . As expeeriments . In addieriments , suggestin vitro toxicity toward a normal cell line compared to the currently used drug cisplatin, and that its IC50 values in cancer cell lines were 20–100 times lower than that of cisplatin. These results prompted the investigation of this drug in an in vivo animal model, which confirmed the anti-tumor activity observed in vitro, indicating the potential of this agent as an anticancer therapy.We previously reported the antitumor effect of dsBPT on a glioblastoma cell line . In the dsBPT was initially used as a chelater to form a gold complex . In factIn addition to chelating gold ion, we found that dsBPT can form a complex with iron, zinc, and copper ions (data not shown). Although the exact target of dsBPT has not been identified, this agent clearly induces apoptosis , which iet al, reported that a copper chelate of thiosemicarbazone NSC 689534 induced oxidative/ER stress and inhibited tumor growth in vitro and in vivo partially through autophagy [We observed that dsBPT did not induce apoptosis at lower concentrations (1–2 μM), rather, it blocked cell cycle progression  by activutophagy . Severalutophagy –31. The 2, cisplatin can induce severe nephrotoxicity [2 show severe signs of neuropathy [Since dsBPT can inhibit cancer growth but cannot cause cell death at a relatively low concentration, we tested whether combining this agent with a cytotoxic agent, such as cisplatin, can induce cancer cell death. Cisplatin is a highly effective antineoplastic drug, but it causes dose-dependent side effects that compromise its therapeutic efficacy. When the administration dose exceeds 50 mg/mtoxicity . Furtheruropathy . One wayS1 FigNo significant cell apoptosis was observed when the concentration 1–2 μM of dsBPT and cisplatin as used in cell cycle analysis were applied to both A549 (A), and Tu212 (B) cell lines compare to vehicle control. However, the combination of the two at the concentration of 1–2 μM induced higher apoptosis than any of the single drugs at the same concentration in both cancer cell lines (A and B). .(PDF)Click here for additional data file.S2 FigdsBPT treatment induced LC3 expression in both A549 and Tu212 cell lines. .(PDF)Click here for additional data file.S3 FigdsBPT with cisplatin synergistically enhanced the inhibitory sensibility of the both drugs as indicated by CIs which are less than 1.0. Figure is generated using CalcuSyn 2.0 software (Biosoft).(PDF)Click here for additional data file.S4 FigNo bodyweight loss was observed when mice were treated with 2mg/Kg dsBPT, cisplatin, and combination of the two agents with the same concentration.(PDF)Click here for additional data file.