Characterizing the Number of Kinesin Motors Bound to Microtubules in the Gliding Motility Assay Using FLIC Microscopy.

Intracellular transport by kinesin motors moving along their associated cytoskeletal filaments, microtubules, is essential to many biological processes. This active transport system can be reconstituted in vitro with the surface-adhered motors transporting the microtubules across a planar surface. In this geometry, the kinesin-microtubule system has been used to study active self-assembly, to power microdevices, and to perform analyte detection. Fundamental to these applications is the ability to characterize the interactions between the surface tethered motors and microtubules. Fluorescence Interference Contrast (FLIC) microscopy can illuminate the height of the microtubule above a surface, which, at sufficiently low surface densities of kinesin, also reveals the number, locations, and dynamics of the bound motors.

Exploratory comparisons between different anti-mitotics in clinically-used drug combination in triple negative breast cancer.

Triple-negative breast cancer (TNBC) constitutes a very aggressive type of breast cancer with few options of cytotoxic chemotherapy available for them. A chemotherapy regimen comprising of doxorubicin hydrochloride and cyclophosphamide, followed by paclitaxel, known as AC-T, is approved for usage as an adjuvant treatment for this type of breast cancer. In this study we aimed to elucidate the role of KIF11 in TNBC progression throughout its inhibition by two synthetic small molecules containing the DHPM core (dihydropyrimidin-2(1H)-ones or -thiones), with the hypothesis that these inhibitors could be an interesting option of antimitotic drug used alone or as adjuvant therapy in association with AC. For this purpose, we evaluated the efficacy of DHPMs used as monotherapy or in combination with doxorubicin and cyclophosphamide, in Balbc-nude mice bearing breast cancer induced by MDA-MB-231, having AC-T as positive control. Our data provide extensive evidence to demonstrate that KIF11 inhibitors showed pronounced antitumor activity, acting in key points of tumorigenesis and cancer progression in in vivo xenograft model of triple negative breast cancer, like down-regulation of KIF11 and ALDH1-A1. Moreover, they didn't show the classic peripheral neuropathy characterized by impaired mobility, as it is common with paclitaxel use. These results suggest that the use of a MAP inhibitor in breast cancer regimen treatment could be a promising strategy to keep antitumoral activity reducing the side effects.

Two strongly linked blocks within the KIF16B gene significantly influence wool length and greasy yield in fine wool sheep (Ovis aries)

BACKGROUND A previous genome-wide association study (GWAS) identified the kinesin family member 16B (KIF16B) as a candidate gene related to sheep wool production. In this work, DNA pool sequencing and SNPscanTM high-throughput genotyping methods were used to detect single-nucleotide polymor phisms (SNPs) in the sheep KIF16B gene. The correlations between the SNPs and wool length and greasy wool yield were systematically assessed. RESULTS Forty-five SNPs were identified and 37 of them were genotyped, including 10 exon mutations, 26 intron mutations, and 1 promoter region mutation. Most of the SNPs were of medium genetic diversity and at Hardy-Weinberg equilibrium (HWE). Among them, 10 SNPs were associated with greasy wool yield and 28 SNPs impact the wool length. Five specific SNPs were found to exert significant effects on the wool length in all body parts analyzed in this study. Furthermore, linkage disequilibrium (LD) analysis was conducted among SNP loci and they were found to be significantly associated with economically important traits. Two strongly linked SNP blocks were identified within these SNPs and they might exert significant impacts on the greasy wool yield and wool length. CONCLUSIONS The identified SNPs exert significant effects on wool production and could be considered as potential DNA markers for selecting the individuals with superior phenotypes

A Novel SLC5A5 Variant Reveals the Crucial Role of Kinesin Light Chain 2 in Thyroid Hormonogenesis.

CONTEXT: Iodide transport defect (ITD) (Online Mendelian Inheritance in Man No. 274400) is an uncommon cause of dyshormonogenic congenital hypothyroidism due to loss-of-function variants in the SLC5A5 gene, which encodes the sodium/iodide symporter (NIS), causing deficient iodide accumulation in thyroid follicular cells. OBJECTIVE: This work aims to determine the molecular basis of a patient's ITD clinical phenotype. METHODS: The propositus was diagnosed with dyshormonogenic congenital hypothyroidism with minimal 99mTc-pertechnetate accumulation in a eutopic thyroid gland. The propositus SLC5A5 gene was sequenced. Functional in vitro characterization of the novel NIS variant was performed. RESULTS: Sanger sequencing revealed a novel homozygous missense p.G561E NIS variant. Mechanistically, the G561E substitution reduces iodide uptake, because targeting of G561E NIS to the plasma membrane is reduced. Biochemical analyses revealed that G561E impairs the recognition of an adjacent tryptophan-acidic motif by the kinesin-1 subunit kinesin light chain 2 (KLC2), interfering with NIS maturation beyond the endoplasmic reticulum, and reducing iodide accumulation. Structural bioinformatic analysis suggests that G561E shifts the equilibrium of the unstructured tryptophan-acidic motif toward a more structured conformation unrecognizable to KLC2. Consistently, knockdown of Klc2 causes defective NIS maturation and consequently decreases iodide accumulation in rat thyroid cells. Morpholino knockdown of klc2 reduces thyroid hormone synthesis in zebrafish larvae leading to a hypothyroid state as revealed by expression profiling of key genes related to the hypothalamic-pituitary-thyroid axis. CONCLUSION: We report a novel NIS pathogenic variant associated with dyshormonogenic congenital hypothyroidism. Detailed molecular characterization of G561E NIS uncovered the significance of KLC2 in thyroid physiology.

Association of kinesin family member 2A with increased disease risk, deteriorative clinical characteristics, and shorter survival profiles in acute myeloid leukemia

This study aimed to explore the correlation of kinesin family member 2A (KIF2A) expression with disease risk, clinical characteristics, and prognosis of acute myeloid leukemia (AML), and investigate the effect of KIF2A knockdown on AML cell activities in vitro. Bone marrow samples were collected from 176 AML patients and 40 healthy donors, and KIF2A expression was measured by real-time quantitative polymerase chain reaction. Treatment response, event-free survival (EFS), and overall survival (OS) were assessed in AML patients. In vitro, KIF2A expression in AML cell lines and CD34+ cells (from healthy donors) was measured, and the effect of KIF2A knockdown on AML cell proliferation and apoptosis in HL-60 and KG-1 cells was detected. KIF2A expression was greater in AML patients compared to healthy donors, and receiver operating characteristic curve indicated that KIF2A expression predicted increased AML risk (area under curve: 0.793 (95%CI: 0.724-0.826)). In AML patients, KIF2A expression positively correlated with white blood cells, monosomal karyotype, and high risk stratification. Furthermore, no correlation of KIF2A expression with complete remission or hematopoietic stem cell transplantation was found. Kaplan-Meier curves showed that KIF2A expression was negatively correlated with EFS and OS. In vitro experiments showed that KIF2A was overexpressed in AML cell lines (KG-1, HL-60, ME-1, and HT-93) compared to CD34+ cells, moreover, cell proliferation was reduced but apoptosis was increased by KIF2A knockdown in HL-60 and KG-1 cells. In conclusion, KIF2A showed potential to be a biomarker and treatment target in AML.

Small interfering RNA for cancer treatment: overcoming hurdles in delivery.

In many ways, cancer cells are different from healthy cells. A lot of tactical nano-based drug delivery systems are based on the difference between cancer and healthy cells. Currently, nanotechnology-based delivery systems are the most promising tool to deliver DNA-based products to cancer cells. This review aims to highlight the latest development in the lipids and polymeric nanocarrier for siRNA delivery to the cancer cells. It also provides the necessary information about siRNA development and its mechanism of action. Overall, this review gives us a clear picture of lipid and polymer-based drug delivery systems, which in the future could form the base to translate the basic siRNA biology into siRNA-based cancer therapies.

Key role of the motor protein Kinesin 13B in the activity of homeodomain-leucine zipper I transcription factors.

The sunflower (Helianthus annuus) homeodomain-leucine zipper I transcription factor HaHB11 conferred differential phenotypic features when it was expressed in Arabidopsis, alfalfa, and maize plants. Such differences were increased biomass, seed yield, and tolerance to flooding. To elucidate the molecular mechanisms leading to such traits and identify HaHB11-interacting proteins, a yeast two-hybrid screening of an Arabidopsis cDNA library was carried out using HaHB11 as bait. The sole protein identified with high confidence as interacting with HaHB11 was Kinesin 13B. The interaction was confirmed by bimolecular fluorescence complementation and by yeast two-hybrid assay. Kinesin 13B also interacted with AtHB7, the Arabidopsis closest ortholog of HaHB11. Histochemical analyses revealed an overlap between the expression patterns of the three genes in hypocotyls, apical meristems, young leaves, vascular tissue, axillary buds, cauline leaves, and cauline leaf nodes at different developmental stages. AtKinesin 13B mutants did not exhibit a differential phenotype when compared with controls; however, both HaHB11 and AtHB7 overexpressor plants lost, partially or totally, their differential phenotypic characteristics when crossed with such mutants. Altogether, the results indicated that Kinesin 13B is essential for the homeodomain-leucine zipper transcription factors I to exert their functions, probably via regulation of the intracellular distribution of these transcription factors by the motor protein.

Truncated Tau Induces Mitochondrial Transport Failure Through the Impairment of TRAK2 Protein and Bioenergetics Decline in Neuronal Cells.

Mitochondria are highly specialized organelles essential for the synapse, and their impairment contributes to the neurodegeneration in Alzheimer's disease (AD). Previously, we studied the role of caspase-3-cleaved tau in mitochondrial dysfunction in AD. In neurons, the presence of this AD-relevant tau form induced mitochondrial fragmentation with a concomitant reduction in the expression of Opa1, a mitochondrial fission regulator. More importantly, we showed that caspase-cleaved tau affects mitochondrial transport, decreasing the number of moving mitochondria in the neuronal processes without affecting their velocity rate. However, the molecular mechanisms involved in these events are unknown. We studied the possible role of motor proteins (kinesin 1 and dynein) and mitochondrial protein adaptors (RhoT1/T2, syntaphilin, and TRAK2) in the mitochondrial transport failure induced by caspase-cleaved tau. We expressed green fluorescent protein (GFP), GFP-full-length, and GPF-caspase-3-cleaved tau proteins in rat hippocampal neurons and immortalized cortical neurons (CN 1.4) and analyzed the expression and localization of these proteins involved in mitochondrial transport regulation. We observed that hippocampal neurons expressing caspase-cleaved tau showed a significant accumulation of a mitochondrial population in the soma. These changes were accompanied by evident mitochondrial bioenergetic deficits, including depolarization, oxidative stress, and a significant reduction in ATP production. More critically, caspase-cleaved tau significantly decreased the expression of TRAK2 in immortalized and primary hippocampal neurons without affecting RhoT1/T2 and syntaphilin levels. Also, when we analyzed the expression of motor proteins-Kinesin 1 (KIF5) and Dynein-we did not detect changes in their expression, localization, and binding to the mitochondria. Interestingly, the expression of truncated tau significantly increases the association of TRAK2 with mitochondria compared with neuronal cells expressing full-length tau. Altogether these results indicate that caspase-cleaved tau may affect mitochondrial transport through the increase of TRAK2-mitochondria binding and reduction of ATP production available for the process of movement of these organelles. These observations are novel and represent a set of exciting findings whereby tau pathology could affect mitochondrial distribution in neurons, an event that may contribute to synaptic failure observed in AD.

New pharmacological findings linked to biphenyl DHPMs, kinesin Eg5 ligands: anticancer and antioxidant effects.

Background: Dihydropyrimidin-2-thiones (DHPMs) are a class of heterocyclic compound which have been intensively investigated mainly due to their anticancer activity as kinesin Eg5 inhibitors. Materials & methods: A library of N1 aryl substituted DHPMs were tested against glioma and bladder cancer cell lines. Quantitative structure-activity relationship (QSAR) investigation was performed in order to identify key elements of DHPMs linked with their antiproliferative effect. The toxicity of most active compounds was investigated using Caenorhabditis elegans as the model. Results & conclusion: DHPMs 9, 13 and 17 have been identified as having improved activity against glioma and bladder cell lines as compared with monastrol. Flow cytometry investigations showed that the new compounds induce cell cycle arrest in phase G2/M and cell death by apoptosis. In addition, compound 13 was able to modulate the reactive oxygen species production in vivo in C. elegans. The biphenyl dihydropyrimidinthiones provided a safety profile in C. elegans.

Fluorescence correlation spectroscopy reveals the dynamics of kinesins interacting with organelles during microtubule-dependent transport in cells.

Microtubule-dependent motors usually work together to transport organelles through the crowded intracellular milieu. Thus, transport performance depends on how motors organize on the cargo. Unfortunately, the lack of methodologies capable of measuring this organization in cells determines that many aspects of the collective action of motors remain elusive. Here, we combined fluorescence fluctuations and single particle tracking techniques to address how kinesins organize on rod-like mitochondria moving along microtubules in cells. This methodology simultaneously provides mitochondria trajectories and EGFP-tagged kinesin-1 intensity at different mitochondrial positions with millisecond resolution. We show that kinesin exchange at the mitochondrion surface is within ~100 ms and depends on the organelle speed. During anterograde transport, the mitochondrial leading tip presents slower motor exchange in comparison to the rear tip. In contrast, retrograde mitochondria show similar exchange rates of kinesins at both tips. Numerical simulations provide theoretical support to these results and evidence that motors do not share the load equally during intracellular transport.

Production of a kinesin-related recombinant protein (Lbk39) from Leishmania braziliensis by Leishmania tarentolae promastigotes and its application in the serodiagnosis of leishmaniasis.

The leishmaniases are multifactorial zoonotic diseases requiring a multidisciplinary One Health approach for diagnosis and control. For leishmaniasis diagnosis, here we describe production of a new recombinant protein based on a kinesin-related gene of Leishmania braziliensis (Lbk39), which shows 59% amino acid identity to the L. infantum homologue. The Lbk39 gene was synthesized, inserted into the pLEXSY-sat2 vector and transfected into L. tarentolae cells by electroporation. Culturing was carried out, and the secreted recombinant protein with a C-terminal histidine tag purified using nickel affinity chromatography on the culture supernatant, yielding a final product at 0.4 mg/mL. An indirect enzyme linked immunosorbent assay (ELISA) was standardised using sera from 74 Brazilian patients with cutaneous leishmaniasis and 11 with visceral leishmaniasis. Optimal ELISA conditions were established for the Lbk39 antigen in comparison with a crude extract from L. braziliensis. The sensitivity, specificity analysis and receiver operating characteristic (ROC) curve were determined with a significance level of 5%. The ROC curve showed a good accuracy with an area under curve (AUC) = 0.967, p < 0.001 (0.941-0.993) for CL patients and an AUC = 100 (100-100) for VL patients. The values of sensitivity and specificity were 88 and 98% for CL and 100 and 100% for VL, respectively. The study showed good production and expression of the target protein and has generated a potential new antigen for the diagnosis of leishmaniasis.

Thermodynamic Efficiency of Somatic Exocytosis of Serotonin.

Through somatic exocytosis neurons liberate immense amounts of transmitter molecules that modulate the functioning of the nervous system. A stream of action potentials triggers an ATP-dependent transport of transmitter-containing vesicles to the plasma membrane, that ends with a large-scale exocytosis. It is commonly assumed that biological processes use metabolic energy with a high thermodynamic efficiency, meaning that most energy generates work with minor dissipation. However, the intricate ultrastructure underlying the pathway for the vesicle flow necessary for somatic exocytosis challenges this possibility. To study this problem here we first applied thermodynamic theory to quantify the efficiency of somatic exocytosis of the vital transmitter serotonin. Then we correlated the efficiency to the ultrastructure of the transport pathway of the vesicles. Exocytosis was evoked in cultured Retzius neurons of the leech by trains of 10 impulses delivered at 20 Hz. The kinetics of exocytosis was quantified from the gradual fluorescence increase of FM1-43 dye as it became incorporated into vesicles that underwent their exo-endocytosis cycle. By fitting a model of the vesicle transport carried by motor forces to the kinetics of exocytosis, we calculated the thermodynamic efficiency of the ATP expenses per vesicle, as the power of the transport divided by total energy ideally produced by the hydrolysis of ATP during the process. The efficiency was remarkably low (0.1-6.4%) and the values formed a W-shape distribution with the transport distances of the vesicles. Electron micrographs and fluorescent staining of the actin cortex indicated that the slopes of the W chart could be explained by the interaction of vesicles with the actin cortex and the calcium-releasing endoplasmic reticulum. We showed that the application of thermodynamic theory permitted to predict aspects of the intracellular structure. Our results suggest that the distribution of subcellular structures that are essential for somatic exocytosis abates the thermodynamic efficiency of the transport by hampering vesicle mobilization. It is remarkable that the modulation of the nervous system occurs at the expenses of an efficient use of metabolic energy.

Lysosome motility and distribution: Relevance in health and disease.

Lysosomes are dynamic organelles, which can fuse with a variety of targets and undergo constant regeneration. They can move along microtubules in a retrograde and anterograde fashion by using motor proteins, kinesin and dynein, being main players in extracellular secretion, intracellular components degradation and recycling. Moreover, lysosomes interact with other intracellular organelles to regulate their turnover, such as ER, mitochondria and peroxisomes. The correct localization of lysosomes is relevant in several physiological processes, including appropriate antigen presentation, neurotransmission and receptors modulation in neuronal synapsis, whereas hepatic lysosomes and autophagy are master regulators of nutrient homeostasis. Alterations in lysosome function due to mutation of genes encoding lysosomal proteins, soluble hydrolases as well as membrane proteins, lead to lysosomal storage diseases (LSDs). Lysosomes containing undegraded substrates are finally stacked and therefore miss positioned inside the cell, leading to lysosomal dysfunction, which impacts a wide range of cellular functions.

Antiproliferative activity of monastrol in human adenocarcinoma (MCF-7) and non-tumor (HB4a) breast cells.

Monastrol is an allosteric inhibitor of the mitotic kinesin Eg5 that exhibits an antiproliferative effect against several cell lines. We investigated the antiproliferative effect of monastrol on human breast adenocarcinoma cells (MCF-7) and mammary epithelial cells (HB4a, non-tumoral). Monastrol treatment decreased cell viability only in MCF-7 tumor cells. Real-time cell growth kinetic analysis showed a decrease in the proliferation of MCF-7 cells exposed to monastrol, while in the HB4a cells, only a concentration of 100 µM was able to induce this effect. In a cell cycle analysis, exposure of MCF-7 cells to monastrol led to an increased population of cells in both the G1 and G2/M phases. In HB4a cells, the proportion of cells in the G2/M phase was increased. Monastrol led to an increased mitotic index in both cell lines. Monastrol was not able to induce cell death by apoptosis in any of the cell lines studied. Gene expression analysis was performed to measure the mRNA levels of cell cycle genes, DNA damage indicator gene, and apoptotic related genes. Treatment with monastrol induced in MCF-7 cells a 5-fold increase in the mRNA levels of the CDKN1A gene, an inhibitor of CDKs related with cell cycle arrest in response a stress stimulus, and a 2-fold decrease in CDKN1C mRNA levels in HB4a cells. These results provide evidence that monastrol has a greater antiproliferative effect on MCF-7 tumor cells compared with non-tumor HB4a cells; however, no selective is observed.

Estudo do potencial efeito inibitório do composto sintético CPO001 sobre a quinesina Eg5 análises in vitro com validação de cálculos em bioquímica quântica

Pterocarpanos representam a maior classe de isoflavonóides, depois das isoflavonase estudos recentes tem revelado que os representantes deste grupo podem agir em alvos específicos da mitose. O presente estudo avaliou o potencial citotóxico, genotóxico e mutagênico, do composto CP001,em célulasnormais e linhagens tumorais humanas, além do efeito inibitório sobre a quinesina Eg5fazendo uso de ensaios in vitro e com cálculos de bioquímica quântica.Os ensaios de citotoxicidade mostraram que o CP001apresentou efeito significativo sobre as linhagens tumorais testadas (HL-60, HCT-116, OVCAR-8, SF-295)com IC50 variandoentre 0,2 e 3,61 μM. Efeito confirmado na curva de crescimento cinético em tempo real, onde o CP001inibiu o crescimento da linhagem OVCAR-8 na concentração de 4 μM, semelhante ao paclitaxel (0,5 μM).Desta forma, a fim de determinar o mecanismo de ação envolvido, uma sequência de experimentos in vitroforam realizados.A avaliação do conteúdo de DNA nuclear foi mensurada em células OVCAR-8, para analisar o efeito do pterocarpano sobre as fases do ciclo celular, revelando que o CP001 é capaz de parar o ciclo celular na fase G2/M, na concetração de 5 μM, e com efeito potencializador, quando colocado em conjunto com o paclitaxel. A parada do ciclo celular na fase G2/M pode estar relacionado a ação do composto na tubulina. O ensaio da polimerização da tubulina foi conduzido e mostrou-se que o CP001 possui velocidade de polimerização (Vma = 80.95 mOD/min), inferior a do paclitaxel (Vmax = 100 mDO/min) e próxima a do monastrol (Vmax = 88.46 mOD/min). Estes dados mostram que a interferência na polimerização da tubulina não é tão significativa quanto aquela apresentada no paclitaxel. A ação em proteínas específicas da mitose foi outra possibilidade testada...

Pterocarpans represented the largest isoflavonoid class after isoflavones and recent studies have revealed that representatives of this group can act on specific targets of mitosis.This study evaluated the potential cytotoxic , genotoxic and mutagenic of the CP001 in tumoral and normal human cell lines. The inhibitory effect in Eg5 kinesin and quantum biochemistry calculation has been peformed as well. Cytotoxicity tests showed that CP001 hads ignificant effect on the tested tumoral lines (HL-60, HCT-116, OVCAR-8, SF-295) with IC50 ranging between 0.2 and 3.61 μ M. Effect confirmed bykinetic growth curve in real time, where the CP001inhibit growth of OVCAR-8 cell line at a concentration of 4 uM , similar to paclitaxel (0.5 mM). Thus, in order to determine the mechanism of action involved a sequence of in vitro experiments were per formed .The assessment of nuclear DNA content was measured in OVCAR-8 cells to examine the effect of pterocarpanon the cellcycle phases , showing that CP001is capable to arrest cell cycle at the G2/ M phase, concentration of 5 uM , and potentiating effect when added in combination with paclitaxel.The cell cycle arrest in G2/M phase may be related to action of the compound on tubulin.The tubulin assay polymerization was conducted and revealed that CP001 has polymerization rate (Vmax = 80.95 mOD / min ) b elow paclitaxel(Vmax = 100 mOD / min) and close tomonastrol ( Vmax = 88.46 mOD / min).These data suggest that interference with tubulin polymerization is not as significant as that shown in paclitaxel.Specific action mitosis proteins was another possibility tested...

Fast axonal transport of the proteasome complex depends on membrane interaction and molecular motor function.

Protein degradation by the ubiquitin-proteasome system in neurons depends on the correct delivery of the proteasome complex. In neurodegenerative diseases, aggregation and accumulation of proteins in axons link transport defects with degradation impairments; however, the transport properties of proteasomes remain unknown. Here, using in vivo experiments, we reveal the fast anterograde transport of assembled and functional 26S proteasome complexes. A high-resolution tracking system to follow fluorescent proteasomes revealed three types of motion: actively driven proteasome axonal transport, diffusive behavior in a viscoelastic axonema and proteasome-confined motion. We show that active proteasome transport depends on motor function because knockdown of the KIF5B motor subunit resulted in impairment of the anterograde proteasome flux and the density of segmental velocities. Finally, we reveal that neuronal proteasomes interact with intracellular membranes and identify the coordinated transport of fluorescent proteasomes with synaptic precursor vesicles, Golgi-derived vesicles, lysosomes and mitochondria. Taken together, our results reveal fast axonal transport as a new mechanism of proteasome delivery that depends on membrane cargo 'hitch-hiking' and the function of molecular motors. We further hypothesize that defects in proteasome transport could promote abnormal protein clearance in neurodegenerative diseases.

Simultaneous silencing of VEGF and KSP by siRNA cocktail inhibits proliferation and induces apoptosis of hepatocellular carcinoma Hep3B cells

BACKGROUND: Vascular endothelial growth factor (VEGF) is involved in the growth of new blood vessels that feed tumors and kinesin spindle protein (KSP) plays a critical role in mitosis involving in cell proliferation. Simultaneous silencing of VEGF and KSP, an attractive and viable approach in cancer, leads on restricting cancer progression. The purpose of this study is to examine the therapeutic potential of dual gene targeted siRNA cocktail on human hepatocellular carcinoma Hep3B cells. RESULTS: The predesigned siRNAs could inhibit VEGF and KSP at mRNA level. siRNA cocktail showed a further downregulation on KSP mRNA and protein levels compared to KSP-siRNA or VEGF-siRNA, but not on VEGF expression. It also exhibited greater suppression on cell proliferation as well as cell migration or invasion capabilities and induction of apoptosis in Hep3B cells than single siRNA simultaneously. This could be explained by the significant downregulation of Cyclin D1, Bcl-2 and Survivin. However, no sigificant difference in the mRNA and protein levels of ANG2, involving inhibition of angiogenesis was found in HUVECs cultured with supernatant of Hep3B cells treated with siRNA cocktail, compared to that of VEGF-siRNA. CONCLUSION: Silencing of VEGF and KSP plays a key role in inhibiting cell proliferation, migration, invasion and inducing apoptosis of Hep3B cells. Simultaneous silencing of VEGF and KSP using siRNA cocktail yields promising results for eradicating hepatocellular carcinoma cells, a new direction for liver cancer treatment.