Host cell phospholipids are trafficked to and then modified by Chlamydia trachomatis.

There is little information on the trafficking of eukaryotic lipids from a host cell to either the cytoplasmic membrane of or the vacuolar membrane surrounding intracellular pathogens. Purified Chlamydia trachomatis, an obligate intracellular bacterial parasite, contains several eukaryotic glycerophospholipids, yet attempts to demonstrate transfer of these lipids to the chlamydial cell membrane have not been successful. In this report, we demonstrate that eukaryotic glycerophospholipids are trafficked from the host cell to C. trachomatis. Phospholipid trafficking was assessed by monitoring the incorporation of radiolabelled isoleucine, a precursor of C. trachomatis specific branched-chain fatty acids, into host-derived glycerophospholipids and by monitoring the transfer of host phosphatidylserine to chlamydiae and its subsequent decarboxylation to form phosphatidylethanolamine. Phospholipid trafficking to chlamydiae was unaffected by brefeldin A, an inhibitor of Golgi function. Furthermore, no changes in trafficking were observed when C. trachomatis was grown in a mutant cell line with a nonfunctional, nonspecific phospholipid transfer protein. Host glycerophospholipids are modified by C. trachomatis, such that a host-synthesized straight-chain fatty acid is replaced with a chlamydia-synthesized branched-chain fatty acid. We also demonstrate that despite the acquisition of host-derived phospholipids, C. trachomatis is capable of de novo synthesis of phospholipids typically synthesized by prokaryotic cells. Our results provide novel information on chlamydial phospholipid metabolism and eukaryotic cell lipid trafficking, and they increase our understanding of the evolutionary steps leading to the establishment of an intimate metabolic association between an obligate intracellular bacterial parasite and a eukaryotic host cell.     

Cell-free reconstitution of Fas-, UV radiation- and ceramide-induced apoptosis.

Cell-free systems are valuable tools for the dissection of complex cellular processes. Here we show that cytoplasmic extracts from cells exposed to anti-Fas antibody or UV radiation contain an activity capable of reproducing morphological changes typical of apoptosis in nuclei added to these extracts, as well as internucleosomal cleavage of DNA and proteolysis of a protein known to be cleaved during the apoptosis of intact cells. Extracts from control cell populations were inactive in this respect. These effects were partly blocked by the addition of purified Bcl-2 protein or a competitive inhibitor peptide of interleukin-1 beta-converting enzyme to the extracts. Furthermore, apoptotic activity was induced in cytoplasmic extracts from untreated cells by the addition of ceramide, a lipid second messenger implicated recently in apoptosis signaling. These extracts should prove highly useful in the dissection of molecular events that occur during apoptosis.     

Quantitative analysis and molecular species fingerprinting of triacylglyceride molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry

Herein we describe a rapid, simple, and reliable method for the quantitative analysis and molecular species fingerprinting of triacylglycerides (TAG) directly from chloroform extracts of biological samples. Previous attempts at direct TAG quantitation by positive-ion electrospray ionization mass spectrometry (ESI/MS) were confounded by the presence of overlapping peaks from choline glycerophospholipids requiring chromatographic separation of lipid extracts prior to ESI/MS analyses. By exploiting the rapid loss of phosphocholine from choline glycerophospholipids, in conjunction with neutral-loss scanning for individual fatty acids, overlapping peaks in the ESI mass spectrum were deconvoluted generating a detailed molecular species fingerprint of individual TAG molecular species directly from chloroform extracts of biological samples. This method readily detects as little as 0.1 pmol of each TAG molecular species from chloroform extracts and is linear over a 1000-fold dynamic range. The sensitivity of individual TAG molecular species to ESI/MS/MS analyses correlated with the unsaturation index and inversely correlated with total aliphatic chain length of TAG. An algorithm was developed which identifies sensitivity factors, thereby allowing the rapid quantitation and molecular species fingerprinting of TAG molecular species directly from chloroform extracts of biological samples.     

Lipidomics: an analysis of cellular lipids by ESI-MS

Recognition of the importance of lipid signaling in cellular function has led to rapid progress in the technology of lipid analysis. Measurements of lipid species changes are central to defining the networks of cell signaling (e.g., receptor activation by hormones or drugs) and lipids are involved in many biochemical and pathological processes. During the last several years our laboratory has focused on developing efficient methods for extraction of glycerophospholipids from biological systems and their detection and identification by mass spectrometry. We analyze phospholipid changes in mammalian cells as a result of a defined ligand stimulation strategy that supports the research questions of the consortium. The improvement of mass spectrometry techniques for phospholipid analysis combined with sophisticated computational methods developed in our group has facilitated simultaneous analysis of hundreds of phospholipid species in mammalian cells. This information is presented as Lipid Arrays (or more precisely as virtual arrays) and allows identification of temporal changes in membrane phospholipid species between two contrasting biological conditions (e.g., unstimulated basal vs. stimulated or as a contrast between normal and disease stages). Using the lipidomics approach, we are able to identify approximately 450 phospholipid species from total membrane extracts and qualitatively measure pattern response changes initiated by cell surface receptors. As such, this approach facilitates the elucidation of the metabolic changes induced by a perturbation in the cell and recognition of patterns of signaling.     

Sensitive profiling of chemically diverse bioactive lipids

Here, we present an improved method for sensitive profiling of lipids in a single high-performance liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry experiment. The approach consists of i) sensitive isocratic elution, which takes advantage of C18 column material that is resistant to increased pH values induced by piperidine, ii) chemometric alignment of mass spectra followed by differential analysis of ion intensities, and iii) semiquantitative analysis of extracted ion chromatograms of interest. A key advantage of this method is its wide applicability to extracts that harbor lipids of considerable chemical complexity. The method allows qualitative and semiquantitative analysis of fatty acyls, glycerophospholipids (such as glycerophosphatidylinositols, glycerophosphatidylserines, and glycerophosphatidylcholines in brain extracts), phosphatidylinositol mannosides, acylated glycerophospholipids, sphingolipids (including ceramides and gangliosides in brain extracts), and, for the first time with ESI, prenols and mycolic acids (MAs). MAs are targets in antimycobacterial therapy, and they play an important immunomodulatory role during host-pathogen interactions. We compared high-resolution mass spectra of MAs derived from Mycobacterium bovis Bacille Camette-Guérin during entry into nonreplicative conditions induced by oxygen deprivation (hypoxic dormancy). Although the overall composition is not drastically altered, there are pronounced differences in individual MAs. alpha-MAs accumulate during entry into dormancy, whereas a subpopulation of keto-MAs is almost entirely eliminated. This effect is reversed upon resuscitation of dormant mycobacteria. These results provide detailed chemical information with relevance to drug development and immunobiology of mycobacteria.     

Dramatic differences in the roles in lipid metabolism of two isoforms of diacylglycerol kinase

Lipid species changes for SV40-transformed fibroblasts from wild-type or from diacylglycerol kinase-epsilon (DGKepsilon) or diacylglycerol kinase-alpha (DGKalpha) knockout mice were determined for glycerophospholipids, polyphosphatidylinositides (GPInsP n ) and diacylglycerol (DAG) using direct infusion mass spectrometry. Dramatic differences in arachidonate (20:4 fatty acid)-containing lipids were observed for multiple classes of glycerophospholipids and polyphosphatidylinositides between wild-type and DGKepsilon knockout cells. However, no difference was observed in either the amount or the acyl chain composition of DAG between DGKepsilon knockout and wild-type cells, suggesting that DGKepsilon catalyzed the phosphorylation of a minor fraction of the DAG in these cells. The differences in arachidonate content between the two cell lines were greatest for the GPInsP n lipids and lowest for DAG. These findings indicate that DGKepsilon plays a significant role in determining the enrichment of GPInsP n with 20:4 and that there is a pathway for the selective translocation of arachidonoyl phosphatidic acid from the plasma membrane to the endoplasmic reticulum. In contrast, no substantial difference was observed in the acyl chain composition of any class of glycerophospholipid or diacylglycerol between lipid extracts from fibroblasts from wild-type mice or from DGKalpha knockout mice. However, the cells from the DGKalpha knockout mice had a higher concentration of DAG, consistent with the lack of downregulation of the major fraction of DAG by DGKalpha, in contrast with DGKepsilon that is primarily responsible for enrichment of GPInsP n with arachidonoyl acyl chains.     

Effect of Dietary Lipid on the Acyl Group Composition of Glycerophospholipids of Brain Endothelial Cells in the Developing Rat

Abstract: The effects of dietary lipid on the acyl group composition of glycerophospholipids of isolated brain endothelial cell fractions have been determined in the developing rat. Rats were fed high-protein diets containing differing dietary linoleic/linolenic acid ratios but having a similar caloric percentage, or were fed a fat-free diet. With a diet supplemented with corn oil (linoleic/linolenic ratio, 47:1), the proportion of acyl groups of the linolenic acid series (n-3) declines in all glycerophospholipid fractions compared with the controls (linoleic/linolenic ratio, 8.5:1), but the proportion of linoleic acid series (n-6) tends to rise. Consequently, the n-6/n-3 ratio of these glycerophospholipids is markedly higher with corn oil than the control diet. N-9 groups (oleic acid series) are consistently lower in proportion. With fat-free diet, the proportion of n-9 groups is higher in these glycerophospholipids than in the controls, but there is no change in the n-6/n-3 ratio. Comparing the changes produced in the adult and the developing cell fraction, the developing cell fraction is more responsive to dietary influence than that of the adult.     

Identification of an inhibitor of caspase activation from heart extracts; ATP blocks apoptosome formation

By revealing the biochemistry of apoptosis it is expected we will both improve our understanding of diseases where apoptosis plays an important role and aid the development of therapies for these disorders. Caspases are a family of proteases whose activity is required for apoptosis. In this study, a cell-free system was used to investigate the mechanism of caspase-9 activation in extracts from heart cells. Unlike extracts from other cell types, heart extracts were found to activate caspases poorly. This could be explained by the low levels of Apaf-1 in heart cells. However, subsequent testing showed that heart extracts contained an inhibitor of caspase activation that could block caspase activation in extracts from different cell types. Subsequent purification of the inhibitor of caspase activation from these extracts identified ATP. Caspase-9 is activated by recruitment into a multi-protein complex, the apoptosome, which then activates downstream caspases that kill the cell. Importantly, size exclusion chromatography showed that ATP inhibits apoptosome formation at physiologically relevant concentrations. Together these data support the hypothesis that intracellular ATP concentration is a critical factor in determining whether an apoptotic stimulus can induce apoptosome formation. Thus, the well described fall in intracellular ATP apoptosis is not an epiphenomenon but may be a pro-apoptotic event contributing to cell death.     

Synaptosomal and brain mitochondrial lipids in hibernating and cold-acclimated golden hamsters

Synaptosomes and mitochondria were isolated from the brains of warm-adapted, hibernating, and cold-acclimated golden hamsters (Mesocricetus auratus). Lipid extracts of these subcellular fractions were prepared and assayed for plasmenylethanolamine (ethanolamine plasmalogen) and cholesterol levels. The ganglioside composition of synaptosomes was also determined. Samples from the hibernating animals showed characteristic changes in lipid composition. These changes include decreases in plasmenylethanolamine levels and a shift in the ganglioside composition toward a higher percentage of the more polar gangliosides. Those animals which were exposed to cold and did not hibernate (cold-acclimated) showed no such changes. Fatty acid analyses of synaptosomal and mitochondrial ethanolamine glycerophospholipids demonstrated a similar trend. Samples from hibernators showed decreases in 16:0, 18:0, and 22:6 (n-3), and increases in 16:1, 18:1, and 20:4 (n-6) fatty acids. No changes were detectable in samples from cold-acclimated animals, indicating that hibernating and cold-acclimated hamsters represent chemically distinct populations.     

Proteomic profiling in an animal model of acute pancreatitis

Acute pancreatitis (AP) is an inflammatory disease of the pancreas, which evolves in approximately 20% of the patients to a severe illness associated with a high mortality rate. In this study, we performed a comparative proteomic analysis of pancreatic tissue extracts from rats with AP and healthy rodent controls in order to identify changes in protein expression related to the pathobiological processes of this disease. Pancreatic extracts from diseased and controls rats were analyzed by 2-DE and MS/MS. A total of 125 proteins were identified from both samples. Comparative analysis allowed the detection of 42 proteins or protein fragments differentially expressed between diseased and control pancreas, some of them being newly described in AP. Interestingly, these changes were representative of the main pathobiological pathways involved in this disease. We observed activation of digestive proteases and increased expression of various inflammatory markers, including several members of the alpha-macroglobulin family. We also detected changes related to oxidative and cell stress responses. Finally, we highlighted modifications of 14-3-3 proteins that could be related to apoptosis regulation. These results showed the interest of proteomic analysis to identify changes characterizing pancreatic tissue damage and, therefore, to highlight new potential biomarkers of AP.     

Cytotoxicity and apoptosis induction of some selected marine bacteria metabolites

AIMS: To study the potential apoptosis effects of cytotoxic marine bacterial metabolites on human HeLa cell line. METHODS AND RESULTS: After HeLa cells were routinely cultured, tetrazolium-based colorimetric assay for cytotoxicity was performed to screen the marine bacteria extracts showing 12 strains active. To find the potential active strain with apoptosis mechanism, a battery of apoptosis assays, including AO/EB staining, TUNEL assay (terminal-deoxynucleotidyl transferase mediated nick end labelling), gel electrophoresis and flow cytometry, were used to determine whether apoptosis was involved in HeLa cell cytotoxicity of marine bacterial extracts. The results indicated that four strains could induce cell shrinkage, cell membrane blebbing, formation of apoptotic body and DNA fragmentation. CONCLUSIONS: Crude extracts of 12 of 153 strains of marine bacteria showed cytotoxic effects with ID50 ranged from 77.20 to 199.84 microg ml(-1), in which eight strains of bacteria were associated bacteria. The metabolites in the strains of QD1-2, NJ6-3-1, NJ1-1-1 and SS6-4 were able to induce HeLa cells apoptosis. Furthermore, the assessment by flow cytometry indicated that the hypodiploid apoptotic cells increased in a time-dependent manner, suggesting that induced apoptosis occurred from 24 h to 48 h after the extracts treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggested that the compounds from fermentation in these four marine bacterial strains could be candidates for developing apoptosis specific anti-tumour agents with lower toxicity. This study indicated that associated marine bacteria could be good source to find cytotoxic metabolites, and some cytotoxic marine bacterial metabolites could have apoptosis mechanisms.     

Myogenic differentiation and lipid-raft composition of L6 skeletal muscle cells are modulated by PUFAs

Lipid composition and fatty acid analysis of the major classes of membrane phospholipids were determined during myogenic differentiation of L6 skeletal muscle cells. The cholesterol to glycerophospholipids ratio decreased during differentiation, both in total (TM) and detergent-resistant membranes (DRM). Analyses of the membrane lipids showed that differentiation had a major impact on the molecular composition of glycerophospholipids. A significant decrease in the concentration of saturated fatty acids was detected in glycerophospholipid classes, and to a lesser extent in sphingolipids, while the concentration of 16:1n-7, 18:1n-7 and 18:1n-9 increased. At the same time, the concentration of long polyunsaturated fatty acid chains decreased in TM and DRM glycerophospholipids, resulting in a lower saturated to unsaturated fatty acid ratio in myotubes as compared to myoblasts. Interestingly, the observed n-3/n-6 ratio was lower in differentiated cell membranes. PUFA supplementation of L6 cells led to an increase in myogenic differentiation correlated to an incorporation of added PUFAs in TM and DRM glycerophospholipids. As expected after n-3 PUFA supplementation, the n-3/n-6 ratio was clearly increased in TM and, surprisingly, this was also the case in isolated DRM. n-3 and n-6 PUFAs significantly and time-dependently increased the phosphorylation of kinase p70S6K1 during myogenic differentiation, revealing the activation of the upstream kinase mTORC1, a major regulator of cell cycle and protein translation. In contrast, PUFAs did not affect the phosphorylation of the kinase Akt, another pivotal regulator of cell metabolism. These results suggest that PUFA supplementation modified the membrane lipid composition and affected the differentiation of L6 cells.     

Crk is required for apoptosis in Xenopus egg extracts.

Apoptosis is essential for the development and homeostasis of multicellular organisms. Recently, a cell-free extract prepared from Xenopus eggs was shown to recapitulate intracellular apoptotic pathways in vitro. While many stimuli have been shown to trigger apoptosis in a variety of cell types, the intracellular signaling pathways involved in apoptosis remain largely unknown. Here we show that addition of a recombinant protein containing the phosphotyrosine binding (SH2) domain from the adaptor protein crk, but not those derived from a panel of other signaling proteins, can prevent apoptosis in the Xenopus egg extract system. Furthermore, immunodepletion of endogenous crk protein from the egg extracts, or addition of anti-crk antisera to these extracts, prevents apoptosis. The ability to undergo apoptosis can be restored to these extracts by addition of recombinant crk protein. These results directly demonstrate that crk participates in apoptotic signaling.     

Effect of Ducrosia flabellifolia and Savignya parviflora Extracts on Inhibition of Human Colon and Prostate Cancer Cell Lines

The goal of this study was to investigate whether Ducrosia flabellifolia and Savignya parviflora methanol extract the have effect on colon and prostate cancer cell lines. Analysis of total content of phenolics and flavonoids of each plant extract was carried out. Cytotoxic effect, cell cycle analysis, induction of apoptosis and gene expression of Bcl-2 and Bax genes were studied. Obtained results indicated that, the plant extracts exhibit growth inhibition of used cancer cell lines and induced apoptosis as well as arresting of cell cycle. At the molecular level, changes in gene expression were detected via qPCR and confirmed by western blotting. The exhibited anticancer potentialities of plant extracts against utilized cancer cell lines are due to its containing bioactive compounds. Further detailed isolation, fractionation and characterization of bioactive compounds are needed.     

细叶卷柏提取物的体外抗肿瘤活性

利用MTT法检测细叶卷柏乙酸乙酯和正丁醇提取物对HeLa细胞生长的抑制作用,利用流式细胞术(FCM)比较不同提取物对细胞凋亡的影响。结果显示:细叶卷柏的乙酸乙酯和正丁醇部位抑制细胞生长和诱导细胞凋亡作用均有明显的剂量依赖性。乙酸乙酯部位的IC50值为1.927μg/mL,正丁醇部位的IC50值为24.600μg/mL。因此,细叶卷柏乙酸乙酯部位的体外抗肿瘤活性相对较强,其次是其正丁醇部位,水提部位相对较弱。细叶卷柏是一种潜在的抗肿瘤药用植物。     

Phytochemical analysis and antileukemic activity of polyphenolic constituents of Toona sinensis

Toona sinensis is a traditional Chinese medicine belonging to the Meliaceae family. The aim of this study was to identify the potential compounds responsible for anticancer activity of T. sinensis. The EtOAc extracts of leaves and woods of T. sinensis inhibited cell proliferation and induced apoptosis in human leukemia HL-60 cells. Our phytochemical research of these extracts led to the isolation of various polyphenolic constituents. The chemical structures were determined by spectroscopic analyses. Among isolates, gallic acid and loropetalin D showed inhibition of cell proliferation and possible induction of apoptosis in these cells. Overall, our results revealed the importance of T. sinensis as a chemopreventive medicinal plant. In addition, an analysis of structure–activity relationship indicated that the number of galloyl groups affects their antileukemic potency.     

Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis

We have developed a cell-free system that induces the morphological transformations characteristic of apoptosis in isolated nuclei. The system uses extracts prepared from mitotic chicken hepatoma cells following a sequential S phase/M phase synchronization. When nuclei are added to these extracts, the chromatin becomes highly condensed into spherical domains that ultimately extrude through the nuclear envelope, forming apoptotic bodies. The process is highly synchronous, and the structural changes are completed within 60 min. Coincident with these morphological changes, the nuclear DNA is cleaved into a nucleosomal ladder. Both processes are inhibited by Zn2+, an inhibitor of apoptosis in intact cells. Nuclear lamina disassembly accompanies these structural changes in added nuclei, and we show that lamina disassembly is a characteristic feature of apoptosis in intact cells of mouse, human and chicken. This system may provide a powerful means of dissecting the biochemical mechanisms underlying the final stages of apoptosis.     

Changes in fatty acid composition during cell differentiation in the small intestine of suckling piglets.

Alterations of phospholipid fatty acid composition in the renewing intestine were studied in the infant piglet. Newborn piglets were fed from birth to 2 weeks of age a concentrated cow's milk which defined a standard supply of dietary fatty acids. Phospholipids were isolated from the whole mucosa, isolated intestinal cells and purified brush border membranes. Intestinal cells were isolated according to their position along the crypt-villus axis and cell phospholipids were extracted at each step of differentiation. Changes in fatty acid composition of cell phospholipids were related to those of lactase activity in the corresponding cell homogenates. In cell phospholipids, the relative content of linoleic and linoleic acids increased about 2-fold from crypt base to villus tip. Substantial contents of alkenylacyl glycerophospholipids (plasmalogens) were found in crypt cell phospholipids and in purified brush border membrane phosphatidylethanolamine (11 and 14% of alkenyl groups by weight of total fatty acids, respectively). The proportion of alkenylacyl glycerophospholipids decreased as cells ascended the villus column and became more differentiated. The results show that fatty acid compositional changes in differentiating cell phospholipids occurred in the immature intestine (before weaning) and suggest that these alterations might be related to the appearance of specific functions.     

Hsp60 accelerates the maturation of pro-caspase-3 by upstream activator proteases during apoptosis

The activation of caspases represents a critical step in the pathways leading to the biochemical and morphological changes that underlie apoptosis. Multiple pathways leading to caspase activation appear to exist and vary depending on the death-inducing stimulus. We demonstrate that the activation of caspase-3, in Jurkat cells stimulated to undergo apoptosis by a Fas-independent pathway, is catalyzed by caspase-6. Caspase-6 was found to co-purify with caspase-3 as part of a multiprotein activation complex from extracts of camptothecin-treated Jurkat cells. A biochemical analysis of the protein constituents of the activation complex showed that Hsp60 was also present. Furthermore, an interaction between Hsp60 and caspase-3 could be demonstrated by co-immunoprecipitation experiments using HeLa as well as Jurkat cell extracts. Using a reconstituted in vitro system, Hsp60 was able to substantially accelerate the maturation of procaspase-3 by different upstream activator caspases and this effect was dependent on ATP hydrolysis. We propose that the ATP-dependent 'foldase' activity of Hsp60 improves the vulnerability of pro-caspase-3 to proteolytic maturation by upstream caspases and that this represents an important regulatory event in apoptotic cell death.     

Characterization and phytochemical constituents of Periploca hydaspidis Falc crude extract and its anticancer activities

The current study aims to investigate the anticancer potential of Periploca hydaspidis extracts against HCCLM3 and MDA-MB 231 cell lines with invasive properties and to identify molecular targets underlying its action mechanism. Cytotoxic screening of plant extracts was done via MTT assay against liver and breast cancer cell lines and GC/MS of the best cytotoxic fraction was performed to identify its chemical composition. Flow cytometry detected apoptosis and cell-cycle changes after drug treatment. The specified cells were studied for migration and invasion potential along with performing western blot analysis of proteins involved in apoptosis, cell-cycle, metastasis, and MAPK (Mitogen-activated protein kinase) cell-signaling pathway. The results revealed the crude methanol (PHM) fraction of P. hydaspidis shown dose and time dependent cell-proliferative inhibition response. GC/MS analysis detected 54 compounds of which fatty acids (29.8%), benzenoids (15.7%), and esters (14.3%) constituted the bulk. The inhibitory effect against cancer cells was linked with cell-cycle arrest at G0/G1 phase, induction of apoptosis, reduced migration and invasion capabilities post treatment. PHM induced apoptosis via downregulation of anti-apoptotic (survivin, B-cell lymphoma Extra-large; BCL-XL, X-linked inhibitor of apoptosis protein; XIAP, Myelocytomatosis; C-myc), metastatic (Matrix metallopeptidases 9/2; MMP9/2), and cell-cycle regulatory (cyclin D1 and E) proteins, whereas upregulation of pro-apoptotic proteins (Bcl-2 homologous antagonist/killer; BAK, Bcl-2-Associate X protein; BAX, cleaved caspases; 3,7,8,9, and PARP) and activation of MAPK (Jun amino-terminal kinase; JNK and P38) pathway. P38 was needed for PHM-induced apoptosis, where the inhibition of P38 by pharmacological inhibitor (SB239063) diminished the apoptotic effects. Overall, our results conclude that PHM can inhibit cell-proliferation and induce apoptotic effects by activation of P38 MAPK cell-signaling pathway. This suggests the methanol fraction of P. hydaspidis (PHM) to have anticancer compounds, potentially useful for treating liver and breast cancer. In future, one-step advance studies of PHM regarding its role in metastatic inhibition, immune response modulation for reducing tumor, and inducing apoptosis in suitable animal models would be an interesting and promising research area.