N-phenyl pyrazoline derivative inhibits cell aggressiveness and enhances paclitaxel sensitivity of triple negative breast cancer cells.

Protein kinase dysregulation induces cancer cell aggressiveness leading to rapid tumor progression and poor prognosis in TNBC patients. Many small-molecule kinase inhibitors have been tested in clinical trials to treat TNBC patients. In the previous study, we found that N-phenylpyrazoline small molecule acts as a protein kinase inhibitor in cervical cancer cells. However, there remains unknown about N-phenyl pyrazoline potency as a kinase inhibitor and its anti-cancer activity in TNBC cells. In this study, we investigated the activity of N-phenyl pyrazoline against TNBC cells via tyrosine kinase inhibition. Based on the MTT assay, the IC50 values for the N-phenyl pyrazoline 2, 5, A, B, C, and D against Hs578T were 12.63 µM, 3.95 µM, not available, 18.62 µM, 30.13 µM, and 26.79 µM, respectively. While only P5 exhibited the IC50 against MDA MB 231 (21.55 µM). Further, N-phenyl pyrazoline 5 treatment significantly inhibited the cell proliferation rate of Hs578T and MDA MB 231 cells. The migration assay showed that treatment with the compound N-phenyl pyrazoline 5 with 4 µM concentration significantly reduced cell migration of Hs578T cells. N-phenyl pyrazoline 5 treatment at 1 µM and 2 µM was able to reduce the tumorsphere size of Hs578t cells. A combination treatment of P5 and paclitaxel showed a synergistic effect with a combination index score > 1 in both TNBC cells. Further, the P5 predictively targeted the protein kinases that significantly correlated to breast cancer prognosis. The GSEA analysis result shows that receptor tyrosine kinase, Notch3, Notch4, and Ephrin signaling pathways were targeted by P5. The P5 treatment reduced the EGFR expression level and activation in TNBC cells.

Pyrazoline B-Paclitaxel or Doxorubicin Combination Drugs Show Synergistic Activity Against Cancer Cells: In silico Study.

Background: Multidrug resistance in various cancer types is a major obstacle in cancer treatment. The concept of a single drug molecular target often causes treatment failure due to the complexity of the cellular processes. Therefore, combination chemotherapy, in which two or more anticancer drugs are co-administered, can overcome this problem because it potentially have synergistic efficacy besides reducing resistance, and drug doses. Previously, we reported that pyrazoline B had promising anticancer activity in both in silico and in vitro studies. To increase the efficacy of this drug, co-administration with established anticancer drugs such as doxorubicin and paclitaxel is necessary. Materials and Methods: In this study, we used an in silico approach to predict the synergistic effect of pyrazoline B with paclitaxel or doxorubicin using various computational frameworks and compared the results with those of an established study on the combination of doxorubicin-cyclophosphamide and paclitaxel-ascorbic acid. Results and Discussion: Drug interaction analysis showed the combination was safe with no contraindications or side effects. Furthermore, molecular docking studies revealed that doxorubicin-pyrazoline B and doxorubicin-cyclophosphamide may synergistically inhibit cancer cell proliferation by inhibiting the binding of topoisomerase I to the DNA chain. Moreover, the combination of pyrazoline B-paclitaxel may has synergistic activity to cause apoptosis by inhibiting Bcl2 binding to the Bax fragment or inhibiting cell division by inhibiting α-ß tubulin disintegration. Paclitaxel-ascorbic acid had a synergistic effect on the inhibition of α-ß tubulin disintegration. Conclusion: The results show that this combination is promising for further in vitro and in vivo studies.

A Potent EGFR Inhibitor, N-Phenyl Pyrazoline Derivative Suppresses Aggressiveness and Cancer Stem Cell-Like Phenotype of Cervical Cancer Cells.

Objective: Metastasis causes approximately 90% of cancer-related deaths, including in cervical cancer patients. Uncontrolled cell proliferation, migration, and cancer stemness act as critical events in primary tumor growth and cancer metastasis progression in cervical cancer. Here, we investigated the anti-proliferative, anti-migration, and cancer stemness inhibition activity of N-phenyl pyrazoline derivatives against cervical cancer cells. Methods: The chalcone and phenylhydrazine were used to synthesize the N-phenyl pyrazoline 2/5 (P2 and P5). The MTT, colony formation, and wound healing assays were performed to evaluate the N-phenyl pyrazoline effect in HeLa cells. The N-phenyl pyrazoline's protein target was predicted using SwissTargetPrediction and AutoDock Vina software. The Western blotting assay was performed to evaluate the target proteins. The public dataset analysis was used to confirm the clinical relevance of target protein in cervical cancer patients. Results: N-phenyl pyrazoline 2 and 5 were successfully synthesized. The N-phenyl pyrazolines 2 and 5 exhibit cytotoxic effect in HeLa cell line with 20.26 µM, 4.708 µM of IC50, respectively. Further study shows that the N-phenyl pyrazoline 5 suppresses the cell proliferation and migration ability of HeLa cell line in a dose-dependent manner. Target prediction and molecular docking reveal that EGFR and ERBB2 protein as the main target of the N-phenyl pyrazoline 5 compound. The N-phenyl pyrazoline 5 suppresses the EGFR expression level but not the total ERK1/2. Public data and GSEA analysis found that the EGFR high expression level is positively associated with poor survival, cancer metastasis-related signaling pathways, and cancer stem cell markers in cervical cancer patients. In addition, the N-phenyl pyrazoline 5 reduces the HeLa's tumorsphere size and cancer stem cell marker, CD133. Conclusion: N-phenyl pyrazoline 5 suppresses the cell viability, proliferation, migration, and cancer stem cell-like phenotype of cervical cancer cells via EGFR inhibition.

Soil-derived Streptomyces sp. GMR22 producing antibiofilm activity against Candida albicans: bioassay, untargeted LC-HRMS, and gene cluster analysis.

Biofilm-forming fungi, Candida albicans, are currently a serious problem in infectious disease cases. Soil bacteria Streptomyces sp. GMR22 have a large genome size and antifungal metabolites against C. albicans, but its potential antibiofilm activity is not clearly defined. The aims of this study were to determine the antibiofilm activity of GMR22 against C. albicans, identify the main constituents of active extracts, and investigate the biosynthesis gene clusters encoding the enzymes related to metabolism pathways. Antifungal and antibiofilm measurements were performed using in vitro assays on C. albicans ATCC 10231. Main constituents of active extracts were analyzed using untargeted Liquid Chromatography tandem High-Resolution Mass Spectrometry (LC-HRMS). RAST software was applied to investigate the gene clusters of the biosynthesis pathways based on whole genome sequences. Chloroform extract of GMR22 has antifungal and antibiofilm properties at 13-420 µg/mL with palmitic acid (C16H32O2, 273.27028 Da), a saturated fatty acid as a major constituent (42.74). Streptomyces sp. GMR22 has 53 subsystems related to fatty acids biosynthesis (Fab) FAS II. The Kyoto Encyclopedia of Gene and Genome map of Fab revealed 10 of 21 (47.6%) gene clusters encode enzymes related to Fab. There were six gene clusters encoding the enzymes related to the hexadecenoic acid (palmitic acid) biosynthesis pathways: 6.4.12; FabD, FabH, FabF, FabG, FabI and 1.14.192. Each enzyme was encoded by 3-14 genes. These results confirmed that soil Streptomyces sp. GMR22 bacterium has remarkable biotechnological potential by producing fatty acids which are mostly palmitic acid as an active antibiofilm agent against C. albicans.

Synthesis and in vitro assay of hydroxyxanthones as antioxidant and anticancer agents.

In the present work, three hydroxyxanthones were synthesized in 11.15-33.42% yield from 2,6-dihydroxybenzoic acid as the starting material. The chemical structures of prepared hydroxyxanthones have been elucidated by using spectroscopic techniques. Afterward, the hydroxyxanthones were evaluated as antioxidant agents through radical scavenging assay; and anticancer agents through in vitro assays against WiDr, MCF-7, and HeLa cancer cell lines. Hydroxyxanthone 3b was categorized as a strong antioxidant agent (IC50 = 349 ± 68 µM), while the other compounds were categorized as moderate antioxidant agents (IC50 > 500 µM). On the other hand, hydroxyxanthone 3a exhibited the highest anticancer activity (IC50 = 184 ± 15 µM) and the highest selectivity (SI = 18.42) against MCF-7 cancer cells. From the molecular docking study, it was found that hydroxyxanthone 3a interacted with the active sites of Topoisomerase II protein through Hydrogen bonding with DG13 and π-π stacking interactions with DA12 and DC8. These findings revealed that hydroxyxanthones are potential candidates to be developed as antioxidant and anticancer agents in the future.

An Update on the Anticancer Activity of Xanthone Derivatives: A Review.

The annual number of cancer deaths continues increasing every day; thus, it is urgent to search for and find active, selective, and efficient anticancer drugs as soon as possible. Among the available anticancer drugs, almost all of them contain heterocyclic moiety in their chemical structure. Xanthone is a heterocyclic compound with a dibenzo-γ-pyrone framework and well-known to have "privileged structures" for anticancer activities against several cancer cell lines. The wide anticancer activity of xanthones is produced by caspase activation, RNA binding, DNA cross-linking, as well as P-gp, kinase, aromatase, and topoisomerase inhibition. This anticancer activity depends on the type, number, and position of the attached functional groups in the xanthone skeleton. This review discusses the recent advances in the anticancer activity of xanthone derivatives, both from natural products isolation and synthesis methods, as the anticancer agent through in vitro, in vivo, and clinical assays.

In Vitro Antiplasmodial, Heme Polymerization, and Cytotoxicity of Hydroxyxanthone Derivatives.

The previous study showed that xanthone had antiplasmodial activity. Xanthone, with additional hydroxyl groups, was synthesized to increase its antiplasmodial activity. One of the strategies to evaluate a compound that can be developed into an antimalarial drug is by testing its mechanism in inhibiting heme polymerization. In acidic condition, hematin can be polymerized to ß-hematin in vitro, which is analog with hemozoin in Plasmodium. This study was conducted to evaluate the antiplasmodial activity of hydroxyxanthone derivative compounds on two strains of Plasmodium falciparum 3D-7 and FCR-3, to assess inhibition of heme polymerization activity and determine the selectivity of hydroxyxanthone derivative compounds. The antiplasmodial activity of each compound was tested on Plasmodium falciparum 3D-7 and FCR-3 with 72 hours incubation period, triplicated in three replications with the microscopic method. The compound that showed the best antiplasmodial activity underwent flow cytometry assay. Heme polymerization inhibition test was performed using the in vitro heme polymerization inhibition activity (HPIA) assay. The antiplasmodial activity and heme polymerization inhibition activity were expressed as the 50% inhibitory concentration (IC50). In vitro cytotoxicity was tested using the MTT assay method on Vero cell lines to determine its selectivity index. The results showed that among 5-hydroxyxanthone derivative compounds, the 1,6,8-trihydroxyxanthone had the best in vitro antiplasmodial activity on both 3D-7 and FCR-3 Plasmodium falciparum strains with IC50 values of 6.10 ± 2.01 and 6.76 ± 2.38 µM, respectively. The 1,6,8-trihydroxyxanthone showed inhibition activity of heme polymerization with IC50 value of 2.854 mM and showed the high selectivity with selectivity index of 502.2-556.54. In conclusion, among 5-hydroxyxanthone derivatives tested, the 1,6,8-trihydroxyxantone showed the best antiplasmodial activity and has heme polymerization inhibition activity and high selectivity.

Bioactivities of Lyngbyabellins from Cyanobacteria of Moorea and Okeania Genera.

Cyanobacteria are reported as rich sources of secondary metabolites that provide biological activities such as enzyme inhibition and cytotoxicity. Ten depsipeptide derivatives (lyngbyabellins) were isolated from a Malaysian Moorea bouillonii and a Red Sea Okeania sp.: lyngbyabellins G (1), O (2), P (3), H (4), A (7), 27-deoxylyngbyabellin A (5), and homohydroxydolabellin (6). This study indicated that lyngbyabellins displayed cytotoxicity, antimalarial, and antifouling activities. The isolated compounds were tested for cytotoxic effect against human breast cancer cells (MCF7), for antifouling activity against Amphibalanus amphitrite barnacle larvae, and for antiplasmodial effect towards Plasmodium falciparum. Lyngbyabellins A and G displayed potent antiplasmodial effect against Plasmodium, whereas homohydroxydolabellin showed moderate effect. For antifouling activity, the side chain decreases the activity slightly, but the essential feature is the acyclic structure. As previously reported, the acyclic lyngbyabellins are less cytotoxic than the corresponding cyclic ones, and the side chain increases cytotoxicity. This study revealed that lyngbyabellins, despite being cytotoxic agents as previously reported, also exhibit antimalarial and antifouling activities. The unique chemical structures and functionalities of lyngbyabellin play an essential role in their biological activities.

CCL2 and IL18 expressions may associate with the anti-proliferative effect of noncontact electro capacitive cancer therapy in vivo.

Background: Noncontact Electro Capacitive Cancer Therapy (ECCT) is a novel treatment modality in cancer. Chemokine (C-C motif) ligand 2 (CCL2) has a major role in the outgrowth of metastatic breast cancer. Interleukin 18 (IL18) plays a role in macrophage alteration, which leads to excessive angiogenesis. This study aims to elaborate on the association of CCL2, IL18, IL23α, and TNF-α (tumor necrosis factor-alpha) expression with the anti-proliferative effect of ECCT in rat breast tumor tissue.   Methods: Low intensity (18 Vpp) and intermediate frequency (150 kHz) alternating current-electric field (AC-EF) between two capacitive electrodes were exposed as external EF to a rat cage. Twenty-four rats were divided into four groups of six replicates. Breast tumor tissues were collected from 7, 12-dimethylbenz[a]anthracene (DMBA)-induced rats. Two groups were non DMBA-induced rats without ECCT exposure (NINT) and with (NIT). The other two groups were DMBA-induced rats without ECCT exposure (INT) and with (IT). Mammary glands and breast tumor tissues were collected from each group and preserved. Hematoxylin-eosin and immunohistochemistry staining were performed on paraffin sections of tissues using anti-PCNA, anti-ErbB2, anti-Caspase3, and anti-CD68. CCL2, IL18, IL23α, and TNF-α mRNA relative expressions were analyzed using qRT-PCR. Results: ECCT exposure may cause the reduction of PCNA protein expression as well as ErbB2 on breast tumor tissues, but it causes the increase of Caspase3 and macrophage CD68 protein. In rat breast tumor tissues of IT groups, the mRNA expression of CCL2 and IL18 are significantly down-regulated, in contrast with the up-regulated expression of these cytokines in tumor tissues of the INT group. IL23α and TNF- α expression remained similar in both groups. Conclusion: CCL2 and IL18 expressions have an association with the inhibition of breast tumor cell proliferation affected by ECCT exposure.

The 1-monolaurin inhibit growth and eradicate the biofilm formed by clinical isolates of Staphylococcus epidermidis.

BACKGROUND: Biofilm is one of the causes of antibiotic resistance. One of the biofilm-producing bacteria is Staphylococcus epidermidis which has been proven to infect long-term users of urinary catheters and implant devices. The 1-monolaurin compound has been known to have an antimicrobial effect. However, its effect on clinical isolates of S. epidermidis in producing biofilm has not been established. This study was conducted to investigate the effect of 1-monolaurin towards biofilm forming clinical isolates of S. epidermidis. METHODS: The experiment used micro broth dilution technique which consists of test group (1-monolaurin), positive control group (rifampicin), solvent group, negative control group (clinical isolate of S. epidermidis), and media group (TSB media). The Minimal Inhibition Concentration (MIC) was determined by incubating bacteria added with 1-monolaurin (1000-1953 µg/mL) or rifampicin (250-0,488 µg/mL) for 24 h. The MIC was determined visually. After that, the incubated bacteria was cultured in TSA media to determine Minimal Bactericidal Concentration (MBC). The assessment of Biofilm inhibitory Concentration (BIC) and Biofilm Eradication Concentration (BEC) was conducted with the same way, the difference was BIC intervened directly with compound meanwhile BEC was incubated for 24 h in 37 °C before the intervention. Then, the specimen was reincubated to grow biofilm at the microplate, washed with PBS and stained with 1% of crystal violet. The optical density (OD) was measured at a wavelength of 595 nm. The percentage of BIC and BEC then were calculated, continued to probit analysis regression to determine the BIC50, BIC80, BEC50, and BEC80. RESULTS: The MIC dan MBC of 1-monolaurin and rifampicin were > 1000 µg/mL, > 1000 µg/mL, ≤0.488 µg/mL, and 1.953 µg/mL respectively. BIC50 and BIC80 of 1-monolaurin and rifampicin were 26.669 µg/mL, 168.688 µg/mL, 0.079 µg/mL, and 0.974 µg/mL respectively. The BEC50 and BEC80 of 1-monolaurin and rifampicin were 322.504 µg/mL, 1338.681 µg/mL, 5.547 µg/mL, dan 17.910 µg/mL respectively. CONCLUSION: The 1-monolaurin can inhibit growth and eradicate the biofilm formed by clinical isolates of S. epidermidis, however, it has neither inhibit nor kill planktonic cells of S. epidermidis.

Monomyristin and Monopalmitin Derivatives: Synthesis and Evaluation as Potential Antibacterial and Antifungal Agents.

In the present work, monoacylglycerol derivatives, i.e., 1-monomyristin, 2-monomyristin, and 2-monopalmitin were successfully prepared from commercially available myristic acid and palmitic acid. The 1-monomyristin compound was prepared through a transesterification reaction between ethyl myristate and 1,2-O-isopropylidene glycerol, which was obtained from the protection of glycerol with acetone, then followed by deprotection using Amberlyst-15. On the other hand, 2-monoacylglycerol derivatives were prepared through enzymatic hydrolysis of triglycerides in the presence of Thermomyces lanuginosa lipase enzymes. The synthesized products were analyzed using fourier transform infrared (FTIR) spectrophotometer, gas or liquid chromatography-mass spectrometer (GC-MS or LC-MS), and proton and carbon nuclear magnetic resonance (¹H- and 13C-NMR) spectrometers. It was found that monomyristin showed high antibacterial and antifungal activities, while 2-monopalmitin did not show any activity at all. The 1-monomyristin compound showed higher antibacterial activity against Staphylococcus aureus and Aggregatibacter actinomycetemcomitans and also higher antifungal activity against Candida albicans compared to the positive control. Meanwhile, 2-monomyristin showed high antibacterial activity against Escherichia coli. The effect of the acyl position and carbon chains towards antibacterial and antifungal activities was discussed.

Acute toxicity tests of antiplasmodial N-alkyl and N-benzyl-1,10-phenanthroline derivatives in Swiss mice.

Five new derivatives of N-alkyl and N-benzyl-1,10-phenanthroline had been shown to inhibit growth in vitro of Plasmodium falciparum FCR3 and in vivo of P. berghei. Acute toxicity tests demonstrated that some of those compounds had wide therapeutic indices. Safety tests of five N-alkyl and N-benzyl-1,10-phenanthroline derivatives were conducted in five groups of Swiss mice by a single intraperitoneal injection with various amounts of the test compounds, with chloroquine as comparison. Signs of toxic effects were observed during 24 hours and observations were continued for 14 days on the surviving mice. Mice were weighed before and after the test period. There were immediate behavioral changes among mice in the high dose group including restlessness, tremor, convulsion and eventually death, which was postulated to be due to the test compounds acting on the nervous system. There was no dose-dependent histopathological changes in the internal organs. Histopathological changes, such as congestion, degeneration and necrosis, were not found. There are no significant differences in mean weight gain among the groups of mice treated with the different compounds and controls. These results indicated that those new N-alkyl and N-benzyl-1,10-phenanthroline antiplasmodial compounds were toxic at high dose, but at non-toxic doses had no effect on weight gain and no histopathological effects on the appearance of internal organs.

Additive in vitro antiplasmodial effect of N-alkyl and N-benzyl-1,10-phenanthroline derivatives and cysteine protease inhibitor e64.

Potential new targets for antimalarial chemotherapy include parasite proteases, which are required for several cellular functions during the Plasmodium falciparum life cycle. Four new derivatives of N-alkyl and N-benzyl-1,10-phenanthroline have been synthesized. Those are (1)-N-methyl-1,10-phenanthrolinium sulfate, (1)-N-ethyl-1,10-phenanthrolinium sulfate, (1)-N-benzyl-1,10-phenanthrolinium chloride, and (1)-N-benzyl-1,10-phenanthrolinium iodide. Those compounds had potential antiplasmodial activity with IC(50) values from 260.42 to 465.38 nM. Cysteine proteinase inhibitor E64 was used to investigate the mechanism of action of N-alkyl and N-benzyl-1,10-phenanthroline derivatives. A modified fixed-ratio isobologram method was used to study the in vitro interactions between the new compounds with either E64 or chloroquine. The interaction between N-alkyl and N-benzyl-1,10-phenanthroline derivatives and E64 was additive as well as their interactions with chloroquine were also additive. Antimalarial mechanism of chloroquine is mainly on the inhibition of hemozoin formation. As the interaction of chloroquine and E64 was additive, the results indicated that these new compounds had a mechanism of action by inhibiting Plasmodium proteases.

In vitro and in vivo antiplasmodial activity and cytotoxicity of extracts of Phyllanthus niruri L. herbs traditionally used to treat malaria in Indonesia.

In endemic areas where malaria is prevalent, medicinal plants are often used to treat malaria. This study was conducted to evaluate the in vitro and in vivo antiplasmodial activity and cytotoxicity of extracts of meniran (Phyllanthus niruri L.) herb traditionally used to treat malaria in Indonesia. Three extracts viz aqueous, methanolic and chloroformic extracts were obtained by maceration of the herbs. A radioactive method was used to evaluate the in vitro antiplasmodial activity of the extracts on chloroquine-resistant (FCR-3) and chloroquine-sensitive (D-10) strains of Plasmodium falciparum. In vitro antiplasmodial activity was expressed by the concentration inhibiting 50% of parasite growth (IC50). Cytotoxicity was estimated on Hela cells and the Cytotoxicity Index (CI = IC50 on HeLa cells/IC50 on FCR-3 strain) was calculated to evaluate the safety of tested extracts. A standard 4-day test on P berghei infected mice was used to evaluate the in vivo antiplasmodial activity of the extracts showing strong in vitro antiplasmodial activity, for both the methanolic and aqueous extracts. The in vivo antiplasmodial activity was expressed by the dose inhibiting 50% of parasite growth (ED50). The IC50 values obtained for these extracts against P. falciparum ranged from 2.3 to 202.4 microg/ml. The methanolic extract was the most active in vitro extract with an IC50 that ranged from 2.3 to 3.9 microg/ml and a CI that ranged from 41.3 to 57.5. This was also the most in vivo active extract with an ED50 of 9.1 mg/kg/d. Further study will be conducted to isolate and purify active compounds presented in the methanolic extract.

In vitro antiplasmodial activity and cytotoxicity of newly synthesized N-alkyl and N-benzyl-1,10-phenanthroline derivatives.

A previous study showed that the 1,10-phenanthroline skeleton was active in vitro against chloroquine-resistant and sensitive strains of Plasmodium falciparum. Based on this skeleton, 8 derivatives of N-alkyl and N-benzyl-1,10-phenanthrolines have been synthesized. This study was conducted to evaluate the in vitro antiplasmodial activity and cytotoxicity of these compounds. The in vitro antiplasmodial activity was tested on two strains of P. falciparum, FCR-3 chloroquine-resistant and D10 chloroquine-sensitive strains, while their cytotoxicity was tested on the Vero cell line. The parasite and cell growth were estimated by hypoxantine-[2,8-3H] uptake after 24- and 72-hour incubation with each compound tested. The control parasite or cell free from any compounds was referred to as having 100% growth. For this radioactive method, the IC50 value showing concentration inhibiting 50% of the parasite growth was determined by probit analysis. The results showed that the highest antiplasmodial activity was observed with (1)-N-benzyl-1,10-phenanthrolinium iodide with the IC50 0.18-0.45 microM, and the IC50 of the compound on Vero cells ranged from 2,582.30 to 7,057.71 microM. The cytotoxic/ antiplasmodial ratio indicates that this compound has high selectivity (10,993 +/- 330.79-38,965 +/- 6,888.27).