BRAF and RAS are the most frequently mutated mitogen-activated protein kinase (MAPK) genes in melanoma. Binimetinib is a highly selective MAPK kinase (MEK) 1/2 inhibitor with clinical antitumor activity in NRAS- and BRAFV600-mutant melanoma. We performed a nonrandomized, open-label phase II study, where 183 metastatic melanoma patients received binimetinib 45 mg / 60 mg twice-daily (BRAF arms), or binimetinib 45 mg twice-daily (NRAS arm). Biomarker analyses were prespecified as secondary and exploratory objectives. Here we report the extent of MAPK pathway inhibition by binimetinib, genetic pathway alterations of interest, and potential predictive markers for binimetinib efficacy. Twenty-five fresh pre- and post-dose tumor sample pairs were collected for biomarker analyses, which included assessment of binimetinib on MEK/MAPK signaling by pharmacodynamic analysis of pERK and DUSP6 expression in pre- vs post-dose tumor biopsies; identification of pERK and DUSP6 expression/efficacy correlations; assessment of baseline tumor molecular status; and exploration of potential predictive biomarkers of efficacy of binimetinib. The postbaseline pERK and DUSP6 expression decreased across all arms; no association between reduced pERK or DUSP6 levels with clinical efficacy was observed. Genetic aberrations were similar to previously reported data on clinical melanoma samples. Genetic pathway alterations occurred predominantly within CDKN2A/B, PTEN, and TRRAP (BRAF-mutation) and CDKN2A/B, TP53, and NOTCH2 (NRAS-mutation). Several patients with BRAF mutations had amplification of genes on chromosome 7q; these patients tended to have shorter progression-free survival than other patients with BRAF-mutant melanoma. Further analysis of genetic alterations, including amplifications of growth factor genes, will determine utility as biomarkers for efficacy. BRAF or RAS. Activating NRAS and BRAF mutations are present in approximately 20% [The MAPK signaling pathway  regulates cellular proliferation, survival, and differentiation and contributes to the pathogenesis of melanoma . Multipltely 20% , 7 and 5tely 20% , 8 of prBRAF-mutated melanoma, including the BRAFV600- specific inhibitors vemurafenib (single agent), and dabrafenib (single agent or in combination with trametinib), and mitogen-activated protein kinase (MEK) inhibitors trametinib (single agent or in combination with dabrafenib) and cobimetinib (in combination with vemurafenib) [A number of therapies that directly target the MAPK pathway have been approved for rafenib) –15. The rafenib) –18. In arafenib) .BRAF or NRAS alone can encounter a number of challenges, including resistance to BRAF inhibitors, which occurs frequently, mostly through reactivation of the MAPK pathway; common paths to resistance include BRAF amplification or alternative splicing and mutations in RAS, MAP2K1, and CDKN2A [Therapies targeting either mutated d CDKN2A , 21, amoBRAF-mutated melanoma, no approved targeted therapies exist for NRAS-mutated melanoma, but positive phase III data on PFS have been reported for a study comparing the efficacy of binimetinib (MEK162) versus dacarbazine in unresectable or metastatic NRAS-mutated melanoma (NEMO study) [In contrast to O study) . BinimetO study) .BRAF-mutated melanoma, both alone [BRAF-mutated melanoma [BRAFV600- or NRAS-mutated advanced melanoma. The efficacy and safety results from an earlier data cut-off of February 29, 2012  have been previously reported [NRAS-mutated melanoma (3 confirmed) and 8 of 41 patients (20%) with BRAF-mutated melanoma (2 confirmed) had a partial response. Binimetinib was the first targeted therapy to show activity in patients with NRAS-mutated melanoma.Binimetinib has shown promising results in th alone  and in cth alone . In addimelanoma . The safmelanoma , 28. Thireported . No patiNRAS- and BRAF-mutated melanoma were analysed as prespecified secondary and exploratory objectives to investigate the extent of MAPK pathway inhibition and further genetic pathway alterations, in order to find potential predictive markers of response to binimetinib. Among others, dual-specificity phosphatase 6 (DUSP6) and phosphorylated extracellular signal-regulated kinase (pERK) are known/predicted biomarkers of MAPK inhibition and could potentially predict the extent of response to treatment with binimetinib [In this study NCT01320085), biomarker data from binimetinib-treated patients with 085, biomimetinib .BRAF mutations were treated: 41 received binimetinib 45 mg twice daily (BID), and 25 received binimetinib 60 mg BID (subsequently reduced to 45 mg BID). A total of 117 patients with NRAS mutations received binimetinib 45 mg BID , a total of 183 patients were enrolled. Sixty-six patients with BRAF- and NRAS-mutated arms for two data cut-off points, 29 February 2012 [Efficacy and safety results for this study have been previously reported for the ary 2012  and 7 Jaary 2012 . BiomarkBRAF-mutated 45 mg subgroup; four and eight pairs, respectively, in the BRAF-mutated 60 mg and NRAS-mutated arms). Fourteen pairs were evaluable for DUSP6 analysis (three pairs each in the BRAF-mutated 45 mg and 60 mg arms and eight pairs in the NRAS-mutated arm).Biomarkers were analyzed to evaluate MAPK pathway inhibition and analysis was undertaken to evaluate on-treatment biomarker expression and frequency of tumor genetic alterations at baseline. Results were compared against melanoma cases in The Cancer Genome Atlas (TCGA) database and in the context of clinical outcomes, where appropriate. Twenty-five fresh, paired  tumor samples were collected for pharmacodynamic biomarker analysis. Fifteen pairs were evaluable for pERK analysis  and P53 . Five BRAF-mutated tumors had broad amplifications on chromosome 7 and five others on chromosome 1, while three NRAS-mutated tumors exhibited amplifications on chromosome 11. Amplifications tended to be associated with shorter progression-free survival (PFS); for example, amplifications in CCND1 or CCND3 occurred only in five NRAS-mutated patients with a PFS shorter than the median (≤ 3.6 months).A weak association between specific mutations or total number of mutations with either measure of efficacy was shown among patients with either BRAF and NRAS, such that they would escape notice when examined individually. Among both BRAF- and NRAS-mutated arms, genetic pathway alterations were observed, predominantly within the cell cycle genes . Among the NRAS-mutated group, 45 of 78 patients (58%) had alterations in the cell cycle genes; 27 of 65 patients (42%) had both alterations within the cell cycle genes and a short PFS (≤ 110 days).Beyond individual genes, pathways or coherent classes of genes were examined, namely the cell cycle and PI3K and P53 pathways as well as epigenetic regulators, transcription factors, and DNA damage response genes. The rationale for these groupings is that genes with parallel or related functions relevant to melanoma may harbor mutually exclusive mutations, as is the case for s Figure . Within BRAF- and NRAS-mutated tumor genetic landscape, and explore the link between genetic pathway alterations and the response to binimetinib. Understanding the biological impact of MEK inhibition, and the predictive value of tumor genetic markers and pathway alterations on response, could be invaluable for optimizing the efficacy of targeted therapy. Pharmacodynamic analyses showed MAPK pathway inhibition by binimetinib on Day 15 through decreased pERK levels and DUSP6 gene expression. These decreases were observed in both BRAF- and NRAS-mutated arms, and were moderate and consistent with observations in other cancer studies with MEK inhibitors, such as the observed suppression of pERK by cobimetinib (GDC-0973) both in vitro and in vivo [The biomarker analyses of this study aimed to describe the biological impact of MEK-targeted inhibition of the MAPK pathway with binimetinib, establish the  in vivo . However in vivo , the MEKBRAF- and NRAS-mutated melanoma was concordant with that reported in TCGA melanoma cases. This observation demonstrated a consistency of genetic alterations in this patient subset with historical samples. This equivalence also contributes to the validation of these biomarker results, leading toward a better understanding of changes that occur within patients with BRAF- and NRAS-mutated melanoma and their predictive value.Overall, the tumor genetic landscape for patients with BRAF-mutated tumors had amplification of genes on the long arm of chromosome 7 (7q); seven patients exhibited MET and/or HGF amplifications, with coamplification occurring in three of them. Five of these seven patients (including the three patients with coamplification) had a PFS that was shorter than the median PFS of 3.5 months for the population with BRAF mutations. Particular genes of high interest on 7q include HGF, MET, EZH2, and SMO. The latter two have been associated with driving the progression of melanoma [HGF and MET form a functional pair since they are cognate ligand and receptor, respectively. Among patients in the NRAS-mutated group, CCND1 or CCND3 amplifications were exclusively seen in five patients with shorter PFS, indicating that constitutive CDK4/6 pathway signaling may lead to resistance. In this regard, a phase Ib/II study (NCT01781572) with binimetinib in combination with the CDK4/6 inhibitor ribociclib (LEE011) in patients with NRAS-mutant melanoma has recently completed, with preliminary data from the initial Phase 1b study suggesting a manageable safety profile and favourable efficacy [NRAS-mutant melanoma [Several patients with melanoma , 35; HGFefficacy . Furthermelanoma . We cannBRAF- or NRAS-mutated melanoma through MAPK pathway inhibition, and genomic profiling highlighted genetic alterations of interest that could be used as potential predictive biomarkers of response to binimetinib. Although all comparisons with patient outcomes for these data are currently observational in nature, they are indicative of the potential predictive use of genetic data in future larger cohorts. Combined with interpretation of biomarker data from other ongoing studies of binimetinib, these data could provide context toward understanding the biological impact of and prediction of response to binimetinib. Of relevance, assessment of potential additional biomarkers of efficacy or safety was incorporated in the aforementioned NEMO trial, in the phase III COLUMBUS trial (NCT01909453) comparing binimetinib plus encorafenib with encorafenib or vemurafenib in patients with BRAF-mutated melanoma, and in the phase II LOGIC-2 trial (NCT02159066) investigating sequential encorafenib/binimetinib combination therapy followed by a combination with targeted agents after disease progression in patients with BRAFV600 -mutated melanoma.Binimetinib showed activity in patients with BRAF or NRAS status was assessed using archival or fresh tumor biopsies either at a local or central laboratory (MolecularMD) and analyzed by a semiquantitative polymerase chain reaction (BRAF) or bidirectional Sanger sequencing assay (NRAS). After patient enrollment, all tumor biopsies assessed at local laboratories were sent to the central laboratory for mutational status confirmation.Details of the study have been published previously . BrieflyThe study was designed, undertaken, and reported in accordance with the Declaration of Helsinki and the ICH Harmonised Tripartite Guideline for Good Clinical Practice. The protocol was approved by an institutional review board, independent ethics committee, or research ethics board at each institution. All patients provided written informed consent before screening and additional consent if participating in the exploratory biomarker analysis.NRAS or BRAF status: binimetinib 45 mg BID or 60 mg BID for BRAF-mutant tumors, or binimetinib 45 mg BID for NRAS-mutant tumors. The 60 mg BID dose of binimetinib for patients with BRAF-mutated tumors was subsequently reduced to 45 mg BID per a protocol amendment following two serious adverse events . Treatment was administered in 28-day cycles. Binimetinib was administered orally (film-coated tablet) BID from Day 1 of Cycle 1 and continuously throughout the study.This study was a nonrandomized, open-label phase II study in which patients were divided into one of three treatment arms according to tumor The primary endpoint was the proportion of patients who achieved an objective response . Secondary endpoints included PFS, time to response, safety, tolerability, pharmacokinetics, and pharmacodynamics. Biomarker analyses, the focus of this manuscript, were prespecified secondary and exploratory objectives, and included: assessment of pharmacodynamic effects of binimetinib on MEK/MAPK signaling by analysis of pERK and DUSP6 gene expression in pre- versus post-dose tumor biopsies; examination of correlations between pERK, DUSP6 expression and efficacy; assessment of the baseline molecular status of the tumors and exploration of potential predictive biomarkers of response to binimetinib.Safety and pharmacokinetic assessments and overall efficacy data from an earlier cut-off were reported previously . BiomarkAnalyses were descriptive and exploratory in nature, and no inferential analysis was performed. Data were summarized with respect to demographic and baseline characteristics and all relevant pharmacodynamics and genetic alteration measurements.


Layer-by-layer self-assembly is an approach to develop an ultrathin film on solid support by alternate exposure to positive and negative species with spontaneous deposition of the oppositely charged ions. This paper summarizes various approaches used for fabrication of layer-by-layer self-assembly as well as their utility to produce various devices. The layer-by-layer technique is basically used for formation of multilayer films. A variety of nanomaterials use it for the modification of films to enhance their resultant durability as well as strength. Studies have shown that many different types of materials can be used for fabrication of multilayers. Recently the layer-by-layer self-assembly technique has also been used for fabrication of gas sensors, hydrogen sensors and solar-based cells. Various methods, such as spin deposition, calcinations, and dry-transfer printing are being used for fabrication of thin films. In this review, the author summarizes the various interesting properties as well as fabrication strategies of layer-by-layer self-assembly. Layer-by-layer self-assembly is a technique used to grow an ultrathin film on solid substrates by ‘flip-flop’ or alternate exposure to positive and negative species with instantaneous deposition of the oppositely charged ions . Since tThe most important requirement for layer-by-layer self-assembly is a suitable substrate which can hold as well as support the assembly that is going to be organized on it . A varieLayer-by-layer self-assembly can be instituted on a large number of substrates. They are as follows:2SO4-HNO3 is applied a priori to the TP electrode in order to activate the surface with an initial charge (negative charge of carboxyl groups). A layer-by-layer self-assembly is formed by dousing the negatively charged TP electrode in a positively charged aqueous solution of PEI (polyethyleneimine). To further balance the positive charges these electrodes are then immersed in aqueous solution containing negatively charged multi-walled nanotubes (MWNT). This procedure allows us to fabricate thin film with minimum effort. Furthermore, unabsorbed materials can be washed off by immersing the dipped electrode into ultrapure water in between subsequent dipping steps.Microbial fuel cells (MFCs) are specially designed devices which convert chemical energy of fuel into electrical energy via catalytic action of electrogenic anaerobic microorganisms -8. The mSimilar methodology can also be applied for the formation of amperometric glucose biosensors based on the nine layers of multilayer films using multi-walled carbon nanotubes, glucose oxidase (GOD – extracted from species of Aspergillus Niger) and gold nanoparticles. Carbon nanotubes have unique mechanical, electrical and chemical properties besides having enormous potential for use as an important component of nanoscale electronic devices. Due to efficiency at higher temperatures  and promotion of higher electron transfer, CNTs are an important component of biosensors , 10.A novel strategy to fabricate amperometric biosensors is found in multilayer thin films via layer-by-layer self-assembly of a gold nanoparticles (GNp)/multi-walled carbon nanotubes hybrid and glucose oxidase (GOD) for the analysis of glucose. A prerequisite for the fabrication of biosensors is the immobilization of enzymes on the immobilization support in order to achieve efficient surface immobilization of the enzyme on the surface of the electrode. The hybrid of nanoparticles and nanotubes was prepared by the immobilization of gold nanoparticles on multi-walled carbon nanotubes (MWCNTs). Usually the self-assembly process is carried out on a pre-treated platinum electrode dipped in a solution of polyallyamine (PAA) for surface activation and followed by subsequent washing with buffer saline. At a later stage, the number of layers to be deposited decides the number of dips into different solutions. Several washing steps also follow in between each dipping. Eventually, layers formed on the multilayer thin films comprise the GOD/GNP/MWCNTs/Pt electrode.2O2 at low employed potential, and is thus used for H2O2 detection and biosensor fabrication [In another fabrication strategy, glucose-based biosensors were developed. In this approach, immobilization of glucose oxidase (GOD) was executed for the resultant fabrication of Prussian blue (PB) glucose biosensors using chitosan and multi-walled nanotubes (MWNTs) as a support. Principally, the process for the fabrication of biosensors involves the adsorption of enzymes or electrolytes from a solution onto the surface of an electrode, either through electrostatic forces or covalent bonding . Chitosarication -16.In the fabrication of these types of biosensor, Prussian blue heightens the sensitivity of biosensors to detect glucose even at low potential. Amperometric glucose-based biosensors are fabricated using multilayer films of chitosan/MWNT/GOD. A negatively charged indium-tin-oxide (ITO)/PB electrode is used as a support on which chitosan, MWNTs and GOD are absorbed alternately by electrostatic interaction with subsequent washing and nitrogen drying in between. The resultant assembly of chitosan/MWNT/GOD is repeated until the desired number of layers is attained (6–7). The concomitant biosensor has several advantages, such as high selectivity, lower detection limit, improved sensitivity and higher affinity for glucose, as well as good stability under optimal conditions .More and more interest is being shown in applications of nanotechnology. Dendrimers are being used as building blocks for the fabrication of molecularly thin multilayer films on the basis of electrostatic layer-by-layer self-assembly -30. Dend31482+ and S2−, PAMAM-CdS nanocomposites with amine-terminated polyamidoamine dendrimers were synthesized [Polyamidoamine- (PAMAM-) based dendrimers have also been used as a template to fabricate PAMAM-CdS nanocomposites, which show stable photoluminescence in various solvents, such as water and methanol -56. By tthesized . Polyamithesized , catalysthesized , fabricathesized , etc. Inthesized -63.Polyamidoamines have positive as well as negative charges on their external surface, which facilitates adsorption of oppositely charged substrates and microspheres by electrostatic forces –65. A sp2O3, and used to check potential applicability. A modified liftoff strategy was used to provide an additional safeguard for the cantilever structures.Since the discovery of carbon nanotubes (CNTs), they have been often tried and used in the development of chemical sensors, stress/strain sensors, biological sensors, scanning probes and field-emission displays. Carbon nanotubes are also an important component of nano electromechanical systems (NEMS) and nano electronic devices -71. One The layer-by-layer self-assembly technique can also used to prepare enzymatic thin films. This electrostatic self-assembly (ESA) method is used for electrostatic adsorption of cationic and anionic polyelectrolytes on charged surfaces for the fabrication of organized ultrathin polymer films , 76. Dif7787Different types of techniques applied to functionalize the glassy carbon (GC) surfaces with a negatively charged layer of 4-phenylacetate groups are covalently graphed onto the glassy carbon surfaces to impart initial negative charge. In another approach, a glucose oxidase (GOD) monolayer is formed by an affinity reaction between a glucose-oxidase-conjugated antibody and adsorbed antigen monolayer. An immunologically active layer is developed on the substrate surface by the deposition of an antigen and antibody, leading to an antigen-antibody reaction.n multilayers were assembled by dipping the GCA/PF and GCB/PF (PF-precursor films) alternately into glucose oxidase and poly solutions prepared in buffer saline. Studies suggest that immunology-based modifications of GC surfaces using a protein monolayer can establish a platform for different electrostatic-enzyme multilayer assemblies [These GCA- and GCB-coated glassy carbon surfaces, after washing, were dipped alternately into either poly (PDDA) or poly(styrenesulphonate) (PSS) solutions formulated in saline buffer. Subsequently (GOD-PDDA)semblies .2 [2[2/TiO2[Anti-reflection (AR) thin films with layers of high and low refractive index are applied in various utilities such as optical materials, display devices, solar cells and glazings -103. Pri2 , 107, Ti2 [2, 109, Si[2[2/TiO2, 111 and[2[2/TiO2. By appl[2[2/TiO2 with mul[2[2/TiO2. pH is a[2[2/TiO2, 116. AtAn electrostatic self-assembly (ESA) technique can be used for protection of cotton fabrics from the harmful effects of UV radiation. There are several advantages associated with ESA processing, such as no constraints on size, shape and topography of the charged substrates to be utilized. Fictionalization can be controlled by applying altering polyelectrolyte solutions or charged substances by simple, eco-friendly and energy-preserving approaches. Various types of organic molecules with positive as well as negative charges can also be integrated by using multilayers via layer-by-layer self-assembly deposition -111.Recently, using the ESA technique, anti-UV multilayer coatings on cationic cotton fabrics were developed. In this approach, three different types of fluorescent brightening agents (FBAs) incorporated with anionic and polyelectrolytes on cotton substrates were used in alternative layer-by-layer self-assembly -122. The123Self-assembled thin films were also formed by sequential deposition of aqueous solution of squarylium (SQ) dye and poly (PDDA) onto a glass substrate. This is an effective example of ultrathin organic multilayer films -136. Squ140Layer-by-layer self-assemblies can be utilised in various devices for different functions.2) nanoparticles are used to develop sensors for measuring frequency shifts as a function of gas concentration and relative humidity [2 nanoparticles is produced by layer-by-layer self-assembly using oppositely charged solution on quartz-crystal microbalance (QCM). A subsequent acidic treatment given to these films is followed by neutral water treatment. This treatment usually breaks ionic bonds of weak electrolytes and thus separates the resultant aggregated TiO2 nanoparticles in the thin film. Other applications of TiO2 nanoparticles also include photo catalysts [Titanium dioxide , known as a cationic assembler, and a free plasmid acting as an anionic assembler were fabricated using layer-by-layer self-assembly to form a multilayer film with gold surface (acting as substrate) . These sDifferent types of multilayers can be fabricated using cationic carbon nanotubes with anionic polyanions or anionic carbon nanotubes modified non-covalently with cationic naphthalenes on their outer walls . A majorElectrostatic interaction has been employed to fabricate multi-walled carbon nanotubes (MWNT) multilayer films to construct the layer-by-layer self-assembly of anionic and cationic MWNTs . The dev2 or SiO2 spherical nanoparticles and halloysite nanotubes were deposited on Kraft softwood fibres through layer-by-layer nano assembly, using them alternately with oppositely charged polyelectrolyte solutions [2 nanoparticles, clays such as montmorillonite and tubular halloysite nanotubes were developed on different supports using one or more layers of these nanoparticles by keeping them together through polyion interlayers. Kraft fibres are usually negatively charged [2, clay minerals and other micro/nano particles is required to provide the necessary opaqueness, brightness and wettability to paper.Multilayers of TiOolutions , 171. Th charged -175. Thep-toluenesulphonate) (TMPyP) and deoxyribonucleic acid from aqueous solution onto quartz substrates results in the formation of complex films [A preferred path for electron transfer is the stacking and overlapping of the n- and pi-electrons of DNA bases . Biologiex films . TMPyP cex films  or Zr(IVex films . Despite4 can be used. Mercaptosulphonic acid can be employed as stabilizer for the preparation of silver nanoparticles [Surface-enhanced Raman spectroscopy (SERS) is a powerful micro-analytical technique used in various fields such as biomedicine , thin-fi186articles . The res2O3 and SiO2 particles can be fabricated on Si support using layer-by-layer self-assembly. This is a new avenue to form uniform and dense Yb2O3-SiO2-coating microstructures. Basically, assemblies of both Yb2O3 and SiO2 particles require application of viscous-flow sintering for fabrication [2O3-SiO2 system can be selected due to its role in explicating and developing environmental barrier coatings (EBCs) for various Si-based materials [2SiO5 and Yb2Si2O7 can be usefully used as coating materials for protection of Si3N4 ceramics in high-temperature turbine environments [In the fabrication of layer-by-layer self-assembly, thickness and uniformity of multilayer particle assembly can only be controlled by depositing one particle layer at a time and then passing a complex-shape substrate through cellular and micro reactors , 195. Murication . This coaterials , 198. Ybronments . Variousronments . Slurry-ronments .Drop-coating technology is used for formation of composite films. These formed films can be used for their ability to promote the electrochemical behaviour of biological as well as environmentally crucial compounds . The limMicro and Nano Electro Mechanical Systems (MEMS/NEMS) represent a turbulent interdisciplinary field that can find application in multiple components in variable portable devices. Incorporation of protective coating is crucial in micro electromechanical systems (MEMS) and nano electromechanical systems (NEMS) to give lower friction and higher anti-wear strength to assure the resultant performance, efficiency and dependability of devices . Langmui2 nanoparticles in multilayer films endows them with multifunctional features. In one strategy an ultra-hydrophobic surface was obtained by coating silica nanoparticles on a micro porous polyelectrolyte multilayer surface [2 nanoparticles capped with Y-amino propyl trimethoxysilane were dipped into polyelectrolyte PAH and PAA multilayer films [To impart specific properties, multiple components can be incorporated in nanocomposite films through layer-by-layer self-assembly, while for time- and cost-efficient fabrication of multilayer films, spin-assisted layer-by-layer self-assembly is used . The int surface . By usiner films .2 gas sensing, dye-sensitized solar cells, stent-assisted gene transfer. Recently developed applications include layer-by-layer self-assembly of anionic and cationic CNTs, nanoparticle coatings on microfibres, layer-by-layer self-assembly of DNA-dye complex films, coatings on nanoparticles and improvement of strength and durability of multilayer films for micro electromechanical systems (MEMS) and nano electromechanical systems (NEMS). With substantial advancements in science and technology and greater availability of information regarding layer-by-layer self-assembly strategies, more and more devices, such as biosensors and electromechanical devices, can be expected to be developed using this technique. More applications for layer-by-layer self-assembly will also be developed.LBL self-assembly is an approach to develop an ultrathin film on solid support by alternate exposure to positive and negative species with impromptu deposition of the oppositely charged ions. This review has summarized various advancements in the development of the layer-by-layer self-assembly technique and its applications. Fabrication of layer-by-layer self-assembly requires various substrates such as metals, glass, and polymers. Each substrate has its own unique application in relation to layer-by-layer self-assembly. Various types of multilayers have been self-assembled by the layer-by-layer approach and used in construction of fuel cells, amperometric biosensors, dendrimer multilayers, thin films, anti-reflection thin films and anti-UV multilayers. Important applications of layer-by-layer self-assembly include gas sensors, e.g., H


Acyrthosiphon pisum, requires its nutritional endosymbiont, Buchnera, for the production of essential amino acids. We hypothesize that key aphid genes that regulate the nutritional symbioses respond to host-plant diet when aphids feed on a specialized  compared to a universal host-plant diet (fava), which vary in amino acid profiles. Using RNA-Seq and whole genome bisulfite sequencing, we measured gene expression and DNA methylation profiles for such genes when aphids fed on either their specialized or universal host-plant diets. Our results reveal that when aphids feed on their specialized host-plant they significantly up-regulate and/or hypo-methylate key aphid genes in bacteriocytes related to the amino acid metabolism, including glutamine synthetase in the GOGAT cycle that recycles ammonia into glutamine and the glutamine transporter ApGLNT1. Moreover, regardless of what host-plant aphids feed on we observed significant up-regulation and differential methylation of key genes involved in the amino acid metabolism and the glycine/serine metabolism, a metabolic program observed in proliferating cancer cells potentially to combat oxidative stress. Based on our results, we suggest that this regulatory response of key symbiosis genes in bacteriocytes allows aphids to feed on a suboptimal host-plant that they specialize on.Microbes are known to influence insect-plant interactions; however, it is unclear if host-plant diet influences the regulation of nutritional insect symbioses. The pea aphid,  When an organism symbiotically lives inside another organism’s cells its cellular metabolic processes often become integrated with its hosts’. Archetypes of these ancient cellular integration events are readily observed in eukaryotic cells as mitochondria and plastids . More thAcyrthosiphon pisum) and its bacterial endosymbiont, Buchnera aphidicola, is one of the best-studied models on nutritional symbioses. In this symbiosis, amino acid pathways of both players are integrated together for the production of essential amino acids . If aphid bacteriocytes can alter these key symbiotic genes in response to feeding on different host-plant environments, aphids can potentially optimize and/or compensate for specialized plant diets that are otherwise unsuitable in nutrient profiles.Here, using RNA-Seq and whole genome bisulfite sequencing we investigate if key aphid genes involved in the regulation of the aphid-SI Materials and Methods for details.See Acyrthosiphon pisum strain (LSR1) was divided into 6 independent sub-lines on either fava  or alfalfa  and reared at the same conditions as described in Buchnera abundance was measured per aphid individual with Real Time quantitative PCR (RT-qPCR)  along with transmission electron microscopy (TEM) . Bacteriocytes of each sub-line were dissected and counted using a light microscope.For RNA-Seq trials, for the same 6 sub-lines pea aphid bacteriocytes and body cells were dissected, and RNA extractions were conducted similar to vs. BODY). To identify host-plant specific differences in bacteriocyte gene expression we compared bacteriocytes of alfalfa feeding aphids (ABAC) to bacteriocytes of fava (FBAC) feeding aphids (ABAC vs. FBAC). Specifically, RNA-Seq paired-end data were mapped and analyzed using the HISAT2 (vs. BODY were identified using the likelihood ratio tests (LRT) based on generalized linear models with DESeq2 . Differentially expressed genes for ABAC vs. FBAC were identified using LRT . Statistical significance of differentially expressed genes were determined with FDR adjusted P ≤ 0.05 and ≥1.5X fold change of the normalized expression values.For both host-plant treatments bacteriocyte gene expression was compared to body cell gene expression in this study similar to vs. BODY) as well as between bacteriocytes of alfalfa feeding aphids and fava feeding aphids (ABAC vs. FBAC) were identified using a generalized linear model-based approach with FDR adjusted P ≤ 0.05 .The HISAT2 (vs. BODY), and between bacteriocytes from aphids feeding on alfalfa and fava (ABAC vs. FBAC). Methylation read data were aligned with Bowtie2 and Bismark, as suggested in SI Materials and Methods for details) .Similar to above for expression data, bacteriocyte methylation data were also compared to body tissues regardless of host-plant type  specialist in the field (Vicia faba) compared to the host-plant they specialize on in the field .In this study, the pea aphid strain (LSR1), which originated as an alfalfa . One aphid sub-line feeding on alfalfa (A2) was significantly greater in mass compared to the other aphid sub-lines . Average bacteriocyte number per one aphid individual was 68  and 63  for the fava and alfalfa treatments, respectively. Moreover, Buchnera abundance did not significantly differ between host-plant treatments based on RT-qPCR . Within a unit area (100 µm2), an average of 21 (N = 7) and 24 (N = 5) Buchnera cells were identified within bacteriocytes of 4th instar fava and alfalfa feeding aphids, respectively  (Table S1). For the BAC vs. BODY comparison we identified 1,904 genes that were significantly up-regulated between bacteriocytes and body cells for both host-plant treatments . We further identified 4,211 genes that were significantly down-regulated between bacteriocytes and body cells for both host-plant treatments . To determine host-plant differences between bacteriocytes (ABAC vs. FBAC) we identified 54 genes that were up-regulated and 101 genes that were down-regulated in bacteriocytes of alfalfa feeding aphids compared to bacteriocytes of fava feeding aphids .To investigate if host-plant diet affects the expression of key symbiotic genes of aphids at the mRNA level in bacteriocytes, we first conducted RNA-Seq on bacteriocytes and other aphid body tissues (BAC vs. BODY comparison we found 10 KEGG pathways significantly enriched in bacteriocytes compared to body cells for both host-plant treatments (Table S3a). The top five KEGG pathways in descending order based on the GSEA enrichment score were glycine, serine and threonine metabolism, glyoxylate and dicarboxylate metabolism, phenylalanine metabolism, pentose phosphate pathway, and the nicotinate and nicotinamide metabolism (Table S3a). We also identified five KEGG pathways that were significantly down-regulated in bacteriocytes compared to body cells for both host-plant treatments (Table S3a). Such KEGG pathways in descending order based on the GSEA enrichment score were hippo signaling pathway, notch signaling pathway, other glycan degradation, phototransduction, and neuroactive ligand-receptor interaction.To characterize the functions of genes differentially expressed in bacteriocytes for each comparison we used GSEA (vs. FBAC) we performed GSEA and identified seven KEGG pathways that were significantly enriched in bacteriocytes of alfalfa compared to fava treated aphids. The seven KEGG pathways in descending order based on the GSEA enrichment score were synthesis and degradation of ketone bodies, vitamin B6 metabolism, aminoacyl-tRNA biosynthesis, pyruvate metabolism, Jak-STAT signaling pathway, lipoic acid metabolism, and the butanoate metabolism (Table S3b).To identify the pathways that were differentially expressed in bacteriocytes between host-plant treatments  were significantly enriched in bacteriocytes of alfalfa feeding aphids when compared to bacteriocytes of fava feeding aphids  is a key enzyme of the GS/GOGAT cycle and recycles ammonia into glutamine  belonged to the glycine/serine metabolism  were up-regulated significantly in bacteriocytes compared to body cells. Also, serine hydroxymethyltransferase (SHMT), which converts serine to glycine, and the bifunctional purine biosynthesis protein (PURH) were up-regulated significantly higher in bacteriocytes compared to body cells. The glycine/serine metabolism also relies on the maintenance of the cofactor tetrahydrofolate (THF). In the bacteriocytes of both treatments THF was maintained by the significant up-regulation of genes in the one carbon pool by folate metabolism, and the production of 5,10-methenyl-THF through the Glycine Cleavage System , gluconeogenesis instead of glycolysis appears to be occurring in bacteriocytes compared to body cells as indicated by the up-regulation of malate dehydrogenase (MDH) and phosphoenolpyruvate carboxykinase (PEPCK)  (For both host-plant treatments (BAC  (PEPCK) . In turner; MPC) .Buchnera symbiosis involves the production of uracil. Buchnera is unable to produce its own uracil .Another key pathway that is important in the aphid-vs. BODY; paired t-test, t = 13.47, df = 5, P < 0.0001)   . The ave 0.0001) . Bacteri 0.0066) .vs. BODY; paired t-test, t = 1.76, df = 2, P = 0.22)    . In cont 0.0005) . The perergenic) .t-test, t = 12.05, df = 11, P < 0.001) (P < 0.001) , (P < 0.001)  , t = 8.5< 0.001) ). Also,  0.001), , (t = 17< 0.001) ).a priori groups: bacteriocytes of alfalfa feeding aphids (ABAC), body cells of alfalfa feeding aphids (ABODY), bacteriocytes of fava feeding aphids (FBAC), and body cells of fava feeding aphids (FBODY) were significantly different from one another in CpG methylation profiles . In the PCA ordination, all body samples clustered tightly together in ordination space away from bacteriocyte samples. Using MRPP, we found that methylated CpG profiles of bacteriocyte samples were significantly different compared to body cell samples , with a significantly higher dispersion of within-group differences for bacteriocyte samples (delta = 0.2759) compared to body cell samples (delta = 0.1772). In accord to the PCA ordination MRPP results indicate that bacteriocyte cells have significantly different methylation profiles compared to body cells, which have more similar distributions to one another, regardless of host-plant treatment (vs. FBAC), fava samples are more heterogeneous in CpG profiles compared to alfalfa samples, with a higher dispersion of within-group differences for FBAC (delta = 0.2810) compared to ABAC (delta = 0.2645)  . These rvs. BODY) (Table S5a). Between bacteriocytes of alfalfa feeding aphids compared to fava feeding aphids a total of 294 CpG sites were differentially methylated significantly (ABAC vs. FBAC). For both comparisons, differential CpG methylation was primarily confined to the gene body regions  (Table S5b) as revealed in other non-mammal animals, which may contribute to gene activation and/or alternative splicing  (Table S5). All 441 genes were up-regulated and hypomethylated in bacteriocytes relative to body cells. We also identified 702 genes that are both differentially methylated and differentially spliced between bacteriocyte and body samples of both alfalfa and fava feeding aphids (BAC vs. BODY) . Furthermore, we identified three genes that were both differentially methylated and differentially expressed significantly between bacteriocytes of alfalfa compared to fava feeding aphids . All three genes were down-regulated and hyper-methylated in alfalfa compared to fava feeding aphid bacteriocytes. These genes were the serine/arginine repetitive matrix 1 gene (LOC100160294), the proton-coupled amino acid transporter 4-like (LOC100159667), and the broad-complex core protein isoforms 1/2/3/4/5-like gene (LOC100167015). We also identified three genes that were both differentially methylated and differentially spliced between bacteriocyte samples from alfalfa and fava feeding aphids : anoctamin-1-like (LOC100167803), formin-binding protein 1-like (LOC100166693), and tumor protein D54-like (LOC100164449).To link the patterns of differential DNA methylation with differential gene expression, we identified 441 genes that were both differentially methylated and differentially expressed significantly between bacteriocyte and body samples for both alfalfa and fava feeding aphids . Genes that were both differentially methylated and differentially spliced between bacteriocyte and body samples (BAC vs. BODY) belong to 16 KEGG pathways (Table S6). These pathways include the bulk movement into cells and digestion (15 genes), the degradation, processing, and transport of RNAs (47 genes), four different signaling pathways including one involved in the immune response (Jak-Stat) (37 genes), protein processing and degradation (20 genes), and the biosynthesis of amino acids (6 genes) (Using GSEA (6 genes) .Buchnera symbiosis and the glycine/serine metabolism were both differentially expressed and differentially methylated between the bacteriocytes and body cells (BAC vs. BODY) (ApGLNT1) that was previously characterized to be important for the regulation of Buchnera’s essential amino acid biosynthesis pathways (PURH and cytoplasmic C-1-tetrahydrofolate synthase) in the one carbon pool by folate pathway were hypo-methylated and up-regulated in the bacteriocytes compared to the body cells (Fourteen key aphid genes associated with the integrated aphid-s. BODY) . In addipathways . Also, tdy cells .Buchnera symbiosis are differentially expressed, spliced, and methylated when aphids feed on a specialized compared to a universal host-plant diet. Our data indicate that this regulatory and epigenetic response to distinct host-plant types that vary in amino acid profiles may play a significant role in modulating the aphid-Buchnera amino acid metabolism when aphids feed on their specialized compared to universal host-plant diet. Moreover, this regulatory response in combination with lower aphid fitness when aphids feed on their specialized compared to universal host-plant diet is consistent with the pea aphid engaging in a compensatory metabolic response when it specializes on a less suitable host-plant. We also identified key aphid genes and pathways involved in the aphid-Buchnera symbiosis that are differentially expressed, spliced, and methylated in both host-plant diets. These results collectively suggest that DNA methylation may play both a conserved (maintenance methylation) and an environmentally induced (de novo methylation) regulatory role in bacteriocytes when aphids feed on host-plant diets that vary in amino acid profiles.In this study, we demonstrated for the first time that key aphid genes involved in the regulation of the aphid-Buchnera cell number the aphid-Buchnera integrative metabolism modulates patterns of bacteriocyte DNA methylation and gene expression in response to its specialized host-plant diet, alfalfa, when compared to its universal host-plant diet, fava. Results from our study that provide evidence for this finding include the following: 1) One aphid enzyme  in the GS/GOGAT cycle was enriched in bacteriocytes of alfalfa feeding aphids compared to fava feeding aphids  The transporter ApGLNT1 is significantly up-regulated in the bacteriocytes of alfalfa feeding aphids compared to fava feeding aphids .  The vitamin B6 pathway was significantly enriched in bacteriocytes of alfalfa feeding aphids compared to fava feeding aphids (Table S3b). Vitamin B6 is an essential cofactor in animals and microbes and plays an important role in the amino acid and carbohydrate metabolism and singlet oxygen resistance  The uracil salvage pathway was found to be significantly enriched in bacteriocytes of aphids feeding on alfalfa compared to fava. These results suggest that more uracil potentially for both Buchnera and/or aphid mRNA biosynthesis is needed for aphid bacteriocytes when they feed on alfalfa compared to fava. 5) Also six genes were both differentially expressed/spliced and methylated between bacteriocytes of alfalfa compared to fava bacteriocytes and are involved in amino acid transport, protein kinase activity, calcium activated chloride channel activity, gene regulation via epigenomic interactions utilizing the POZ zinc finger domain, and unknown function. More information on how these genes may help regulate the integrated aphid-Buchnera metabolism when aphids feed on different host-plants is needed. Collectively, these results suggest that when aphids feed on their suboptimal, specialized host-plant, alfalfa, key aphid genes involved in the regulation of the integrative amino acid metabolism are enriched in bacteriocytes.Here we reveal that instead of regulating bacteriocyte or g aphids . The GS/lutamine . 2) The g aphids . Interessistance . CollectBuchnera for the production of essential amino acids.The observed regulatory changes in bacteriocytes are indicative of a compensatory response of aphids feeding on alfalfa that require more essential amino acids compared to aphids on fava. For example, previous amino acid concentration data shows a limitation of essential amino acids in alfalfa sap compared to fava sap . Specifii.e., the Warburg effect, where cellular energy production relies heavily on aerobic glycolysis  gene of Apis melifera increased the expression level of LAM (The genome-wide CpG methylation patterns of the pea aphid showed higher levels of methylation in gene bodies, especially in alfalfa feeding bacteriocytes , which il of LAM .The effect of host-plant treatment on DNA methylation patterns within pea aphid bacteriocytes was evident in our study. For example, our results reveal that the CpG methylation profiles of fava feeding aphid bacteriocytes were more heterogeneous between biological replicates compared to alfalfa feeding bacteriocytes . Such heBuchnera are differentially expressed/spliced, and some of these are differentially methylated. Future studies are needed to investigate how and if these methylated sites can influence the regulation of the bacteriocyte, be inherited, and ultimately drive host-plant specialization broadly in sap-feeding insect-nutritional symbioses. We hypothesize that this host-plant induced metabolic modification to the aphid’s integrative metabolism may ultimately allow aphids to utilize host-plant diets that were once unsuitable.In summary, our findings indicate that when aphids feed on their specialized host-plant, alfalfa, key aphid genes that are involved in the integrative metabolism with


Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells characterized by their immunosuppressive functions. MDSCs expand during chronic and acute inflammatory conditions, the best described being cancer. Recent studies uncovered an important role of MDSCs in the pathogenesis of infectious diseases along with sepsis. Here we discuss the mechanisms underlying the expansion and immunosuppressive functions of MDSCs, and the results of preclinical and clinical studies linking MDSCs to sepsis pathogenesis. Strikingly, all clinical studies to date suggest that high proportions of blood MDSCs are associated with clinical worsening, the incidence of nosocomial infections and/or mortality. Hence, MDSCs are attractive biomarkers and therapeutic targets for sepsis, especially because these cells are barely detectable in healthy subjects. Blocking MDSC-mediated immunosuppression and trafficking or depleting MDSCs might all improve sepsis outcome. While some key aspects of MDSCs biology need in depth investigations, exploring these avenues may participate to pave the way toward the implementation of personalized medicine and precision immunotherapy for patients suffering from sepsis. Sepsis is one of the leading causes of preventable death. Sepsis is defined as a “life-threatening organ dysfunction caused by a dysregulated host response to infection” . The morInnate immune cells, such as monocytes/macrophages, dendritic cells (DCs), and neutrophils, sense microbial and danger-associated molecular patterns  through pattern recognition receptors (PRRs). PRRs are grouped into five main families: toll-like receptors (TLRs), NOD-like receptors, C-type lectins, scavenger receptors, RIG-I-like receptors, and intra-cytosolic DNA sensors . The intDuring the last decades, early goal-directed therapy decreased early mortality from sepsis, which contributed to shift the sepsis ICU population toward a population suffering from chronic critical illness (CCI). Indeed, a subset of ICU patients surviving sepsis develop CCI characterized by long-lasting immunosuppression associated with a persistent, low-grade, inflammation maintained by the continuing release of DAMPs. The underlying inflammation is associated with catabolism and malnutrition. The term persistent inflammation-immunosuppression and catabolism syndrome (PICS) has been proposed to characterize this degraded state. PICS is associated with long-term morbidity, late multiple organ failures and late mortality –11.Clinical trials testing adjunctive therapy to dampen inflammation-related dysfunctions in sepsis have not been conclusive . SeveralWe poorly understand what is responsible for a dysregulated host response and the delay returning to homeostasis in sepsis patients –8, 18. GMDSCs are immature myeloid cells that expand during chronic and acute inflammatory conditions. The premises of MDSC discovery date back more than a century when tumor progression was associated with extra-medullary haematopoiesis and neutrophilia. In the mid-1960s, Lappat and Cawein reported that subcutaneously transplanted A-280 tumor cells generate factors involved in a leucocytosis response that sustains tumor growth . SubsequMDSCs are defined primarily by their immunosuppressive functions. Within sepsis, one may predict that MDSCs play a dual role depending on disease progression. On the one hand, MDSCs may be beneficial by limiting hyper-inflammation during the early stages of sepsis, hence protecting from early organ dysfunction. On the other hand, MDSCs may be detrimental by amplifying long-term immunosuppression and contributing to CCI and/or PICS , 10. As + CD11b+ cells . PMN-MDSCs are CD11b+ Ly6G+ Ly6Clow cells and M-MDSCs CD11b+ Ly6G− Ly6Chigh cells. In humans, PMN-MDSCs are CD11b+ CD14− CD33+ (CD15+ or CD66+) cells and M-MDSCs CD11b+ CD14+ HLA-DRlow/− CD15− cells. PMN-MDSCs overlap phenotypically with mature neutrophils but contrary to PMNs, MDSCs sediment within the PBMC fraction in ficoll gradients after density separation of whole blood. Whether low density gradient (LDGs) PMNs and PMN-MDSCs are the same entity is unclear, albeit the terms is used interchangeably in the literature. The identification of PMN-MDSCs by density gradient is further limited by the rise of not only low-density neutrophils, but also high-density CD62Ldim neutrophils that suppress T cells in the blood of healthy humans infused with endotoxin  and eosinophilic MDSCs (eo-MDSCs) , 35. In Adding to the above caveats, improper cell separation through density gradient and freezing whole blood or PBMC samples before flow cytometry analyses affects the detection of MDSCs, especially PMN-MDSCs. Hence, an objective of future studies is to optimize and harmonize sample handling and flow cytometry strategies  to quantify MDSCs in whole blood. This will facilitate the comparison of results from different studies to determine whether MDSCs are reliable disease biomarkers , 42. StrHematopoietic stem cells differentiate into common myeloid progenitors giving rise to immature myeloid cells. An inflammatory environment, as observed in sepsis, stimulates the egress of immature myeloid cells from the bone marrow into the blood stream and the gain immunosuppressive functions , 44 Fig. The ide3CSK4 (a TLR1/2 agonist) differentiate into immunosuppressive macrophages producing IL-10 , glycoprotein 130 (gp130) and nuclear factor I A  control the expansion and the immunosuppressive functions of MDSCs . MyD88 id to CLP , 58, 59.of IL-10 , 60. Durof IL-10 , 62. In survival . Recent otoxemia .+ CD11b+ MDSCs or PMN-MDSCs harvested from septic donor-mice into recipient mice protects the later from acute endotoxemia, rapidly lethal CLP and Pseudomonas airway infection . Interestingly, the transfer of early MDSCs increases while the transfer of late MDSCs decreases or does not change mortality , have been implicated in the development of MDSCs with different outcomes . For exaMDSCs suppress the activity of immune cells through various mechanisms involving the degradation of L-arginine, the production of reactive oxygen and reactive nitrogen species , the secretion of anti-inflammatory/immunosuppressive cytokines like IL-10 and transforming growth factor (TGF)-β and the activation of T regulatory cells (Tregs) .L-arginine becomes a semi-essential amino acid during sepsis because of increased usage and reduced production. L-arginine shortage is sustained by the production by MDSCs of arginase that metabolizes L-arginine into L-ornithine and urea . L-arginE. coli and group B streptococci (Splenic MDSCs harvested from CLP mice early (3–5 days) and late (10 days) after sepsis onset inhibit T cell proliferation. Early MDSCs secrete less S100A9 than late MDSCs  and, in ptococci  and cleaptococci . Thus, Mptococci , 65. MorMDSCs make up an important proportion of immature myeloid cells. Thus, we will discuss reports analyzing immature granulocytes (IG) in adult sepsis and then move forward to studies that used more elaborated immuno-phenotyping strategies to identify MDSCs. Accumulation of immature myeloid cells is one of the criteria established more than 25 years ago to characterize SIRS (systemic inflammatory response syndrome) and sepsis . The assFew reports demonstrate the immunosuppressive functions of immature myeloid cells in relation with sepsis and/or monitor MDSCs subpopulations using advanced flow cytometry. Since cell preparation  and flow cytometry strategies are not standardized, the phenotype of MDSCs, PMN-MDSCs and M-MDSCs differs between studies .in vitro, suggesting that they represent PMN-MDSCs  and M-MDSCs (SSClow CD14+ CD11b+ CD16− CD15+) does not differ between non-infectious critical ill patients and sepsis patients  representing more than 36% of WBC in ICU patients sampled within 3 days of study inclusion predicts the subsequent occurrence of nosocomial infections  are enriched during gram-negative sepsis, but PMN-MDSCs (CD14− CD15+ LDG) do not differ according to the gram of the causative bacteria  would positively influence patient outcome. Interestingly, more than 30 clinical trials are running targeting MDSCs directly or indirectly in cancer patients . If everIS and TR conceived and structured the manuscript. IS drafted the manuscript and the figure. CT revised the manuscript. TR finalized and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Various physiological processes involve appropriate tissue developmental process and homeostasis - the pathogenesis of several diseases connected with deregulatory apoptosis process. Apoptosis plays a crucial role in maintaining a balance between cell death and division, evasion of apoptosis results in the uncontrolled multiplication of cells leading to different diseases such as cancer. Currently, the development of apoptosis targeting anticancer drugs has gained much interest since cell death induced by apoptosis causes minimal inflammation. The understanding of complexities of apoptosis mechanism and how apoptosis is evolved by tumor cells to oppose cell death has focused research into the new strategies designed to induce apoptosis in cancer cells. This review focused on the underlying mechanism of apoptosis and the dysregulation of apoptosis modulators involved in the extrinsic and intrinsic apoptotic pathway, which include death receptors (DRs) proteins, cellular FLICE inhibitory proteins (c-FLIP), anti-apoptotic Bcl-2 proteins, inhibitors of apoptosis proteins (IAPs), tumor suppressor (p53) in cancer cells along with various current clinical approaches aimed to selectively induce apoptosis in cancer cells. Several death receptors (DRs) such as FAS and TNFR that are known to induce apoptosis also induce necroptosis in different cell types.25 However, programmed necrosis has been seen only under a specific condition when apoptosis has been chemically or genetically repressed or blocked.16 Moreover, another form is apoptosis-like programmed cell death describe the type of cell death which involve apoptotic features, but the cell death occurs in a caspase-independent manner.26Apoptosis typically distinguished from necrosis, which was assumed to represent an opposite way of an unordered cellular explosion in response to severe and irresistible trauma. Interest in non-apoptotic forms of programmed cell death is gradually increasing as more information on this type of cell death is collected.28 Necrosis morphologically characterized by the swelling of cytoplasm and organelles (endoplasmic reticulum and mitochondria), the disruption of the plasma membrane leading to the release of cellular components and cell lysis.29 Cell death by necrosis linked to chronic inflammation, caused by necrosis could enhance the proliferation of tumors.30 However, there is another perception that necrosis could also be programmed in nature as well. Programmed necrosis has been seen only under a specific condition in which apoptosis is chemically or genetically repressed or inhibited.31 Various anti-apoptotic proteins of the Bcl-2 family have been shown to inhibit both apoptotic and necrotic. Intracellular ATP depletion can switch an apoptotic response to a necrotic one. Hence, apoptosis and necrosis are not necessarily independent pathways. Instead, they may share some common messengers, activators, and inhibitors.32Necrosis might happen during several diseases including cancer, neurodegenerative and autoimmune diseases. Necrosis traditionally considered as random, uncontrolled process which is usually initiated by certain stimuli like toxic trauma or physical damage.karkinos” which means carcinoma.33 Cancer defined as an uncontrolled growth of the cell in an abnormal manner which alters the structure of surrounding tissues.34 Cancer cell genotypes demonstrate seven essential alterations in cell physiology which leads to its progression and metastasis.3536The word cancer was first named by a physician Hippocrates around 460-370 B.C. and originated from a Greek word “38 The induction of apoptosis in cancer cells and limit concurrent death of normal cells is the primary objective of cancer therapy.39 Some proteins have been studied to exert pro- and anti-apoptotic activity in the cell and the proportion of these proteins plays an essential part in the regulation of cell death.40 Similarly, the induction of cancer apoptosis is among the main approaches in cancer gene therapy or immunotherapy. The apoptotic inducer, mediator or executioner gene is routinely incorporated in cancer cells to reverse the deficiency of its endogenous counterpart.42 Understanding the cascade of events that trigger apoptosis in response to chemotherapy, and dysfunction of the apoptosis process give insight toward the novel effective therapeutic approach to the development of molecular-targeted specific therapies against cancer.Recent advances in cancer research are focused on the development of new drugs that halt the escape behavior of cancer cells via the execution of apoptosis. To this context, novel apoptotic inducers or sensitizers have been used with the combination of current drugs. Defects in the apoptosis-inducing pathways can eventually lead to the multiplication of neoplastic cells. The resistance of apoptosis stimulates aberrant cellular multiplication which eventually leads to tumorigenesis and is a significant hurdle to active cancer treatment.15 Both of these apoptotic pathways, extrinsic and intrinsic pathways might be lead to same terminal (execution pathway).40The mechanism of apoptosis mainly consist of two core pathways involved in inducing apoptosis; extrinsic pathway and intrinsic pathway. Extrinsic pathway refers to DR-mediated pathway, and the intrinsic pathway is a mitochondrial-mediated pathway.44 This triggering of DRs by specific death ligands (DLs) results in the formation of a death-inducing signaling complex (DISC).12 This DISC consists of the DD-containing Fas-associated death domain (DD) as an adaptor molecule, procaspase-8, procaspase-10, and the cellular FLICE inhibitory proteins (c-FLIPs). The caspase 8 activate in such a manner that prodomain of caspase 8 remains at the DISC, while active caspase 8 dissociates from the DISC to start the cascade of caspase activation which constitutes the execution phase of apoptosis.45 Experimental evidence shows the excessive role of caspases in apoptosis.47 Caspases are essential initiators and executioners of the apoptosis, and their function is very closely related to its structure having different substrate preferences. Some caspases have long pro-domains which involve particular motif like the death effector domain (DED), and caspase recruitment domains (CARD), which allow interacting with other proteins, and connect with signaling pathways. DED includes caspase-8 and caspase-10 while CARD involves caspase-1, caspase-2, caspase-4, caspase-5, caspase-9, caspase-11 and caspase-12.48 Caspases traditionally classified as initiator and effector or executioner caspases by their position in apoptotic signaling cascades.Apoptotic signaling through the extrinsic pathway engaged when extracellular ligands such as TNF (tumor necrosis factor), Fas-L (Fas ligand), and TRAIL (TNF-related apoptosis-inducing ligand) are attached to the extracellular domain of the DR (transmembrane receptors), i.e., the type 1 TNF receptor (TNFR1), Fas  and TRAIL receptors. The order of events involved in the extrinsic phase of apoptosis well characterized by the FasL/FasR and TNF-α/TNFR1 models.5051 Bcl-2 and Bcl-xL (Bcl-2 family member) are anti-apoptotic proteins which prevent the release of cytochrome c.52 The cytochrome c combines with Apaf–1 and procaspase-9 to produce apoptosome. Apoptosome is a multi-protein complex which comprised of a seven-spoke ring-shaped complex, which triggers caspase 9 followed by the activation of caspase-3 signaling caspase cascade which leads to the demolition of cells and ends up to apoptosis.53 Proteins that are generally involved in intrinsic pathway include SMAC/DIABLO , Caspase-9 (Cysteinyl aspartic acid-protease-9), Bcl-2 (B-cell lymphoma protein 2), Bcl-w (Bcl-2 like protein), Nox (Phorbol-12-myristate-13-acetate-induced protein 1), Aven (Cell death regulator Aven) and Myc (Oncogene Myc).15 The dysfunctional mitochondrial results in loss of inner mitochondrial membrane potential, hyperproduction of superoxide ions, disturbance in mitochondrial biogenesis, the outflow of matrix calcium glutathione and release of membrane proteins,15 hold promising potential for cancer therapeutic strategies via induction of apoptosis in cancer cells which are discussed later in this review.The intrinsic pathway refers to mainly mitochondrial-mediated apoptotic pathway. The intrinsic pathway triggered by various extra and intra-cellular stresses, which include oxidative stress, irradiation, and treatment with cytotoxic drugs.54 Caspase-8, and 9 are initiator caspases while caspase-3, caspase-6 and caspase-7, Caspase-10, CAD (Caspase-activated DNAse) and PARP (Poly (ADP-ribose) polymerase) are classified as effector or executioner caspases.56 Initiator caspases activated as a result of autocleavage, which further activates executioner caspases which later proteolyze some substrates leading to apoptosis. They possess long pro-domains that connect large adapter molecules promoting multimerization and result in other caspases activation. However, effector caspases possess short pro-domains which execute apoptosis when activated by initiator caspases. Executioner caspases activate cytoplasmic endonuclease which causes chromatin condensation, the formation of cytoplasmic blebs and apoptotic bodies. Caspases regulate apoptotic cell death via cleavage of numerous target proteins.58 The pathway begins with the activation of execution caspases which further activates cytoplasmic endonuclease. Cytoplasmic endonuclease degrades nuclear material, and proteases followed by the degradation of nuclear and cytoskeletal proteins. Among all executioner caspases, caspase-3 is the most important, and any of the initiator caspases can activate it. Endonuclease CAD is activated explicitly by caspase-3, which causes degradation of chromosomal DNA within nuclei and condensation of chromatin. Execution caspases play an essential role in the cytoskeletal reorganization and formation of cytoplasmic blebs and apoptotic bodies.46Both extrinsic and intrinsic pathways converge at the same point (execution phase). Execution phase refers to the final pathway of apoptosis.59 Death ligands and their respective receptors comprise of TNF-TNFR1, FasL/CD95L-Fas, TWEAK(Apo3L)-TRAMP, TRIAL(Apo2L)-TRAIL-R1 and TRADD-DR6 as shown in 61DRs are associates of TNF superfamily and initiate apoptotic signals when similar death ligands bind to the particular cell surface of DRs.62 A number of HDAC inhibitors like suberoylanilide hydroxamic acid (SAHA), trichostatin A (TSA), LAQ824 (a cinnamic acid hydroxamate), m-carboxycinnamic acid bishydroxamide (CBHA) and MS-275 used in order to stimulate the expression of TRIAL-R1 and TRIAL-R2.64 MG132 is an example of proteasome inhibitors which effectively enhance the expression of TRIAL-R2 (DR5) while PS-341 is another proteasome inhibitor which promotes the expression of TRIAL-R1/2 without affecting the expression of pro-apoptotic Bcl-2 family proteins, cFLIP and caspases.66 A variety of antibodies have been utilized to target DRs (TRIAL), Apo2L/TRAIL showed as a potential cancer therapeutic agent, induce apoptosis in cancer cells via its two major cell DRs TRAIL-R1 and TRAIL-R2.68 They have specifically shown to expressed at higher levels in solid tumors.69 Owing to the ability of TRAIL receptors of inducing cell death specifically in cancer cells, agonistic antibodies against TRAIL receptors have been developed and demonstrated to trigger apoptosis in a number of cancer cells.70 various agonists targeting DRs in clinical trials. Dulanermin targets both TRIAL-R1/2 for colorectal cancer CRC and non-small cell lung cancer NSCLC, Mapatumumab target TRIAL-R1 for advanced solid tumors and NSCLC, PR095780 for Advanced solid tumors, NHL in I and II phase.76 Lexatumumab (HGS-ETR2) and Conatumumab (AMG-655) target TRIAL-R2 for Advanced solid tumors in I phase.80 Despite all the success of TRAIL targeted cancer therapy, TRAIL resistance is a common hindrance in TRAIL-based therapy that restricts the efficacy of these drugs.81DRs play a significant role in the extrinsic apoptotic pathway and therefore emerged as a potential cancer therapeutic target. A variety of agents proposed in order to stimulate the apoptotic function of DRs and ligands in the extrinsic pathway, such as DNA damaging chemotherapeutic agents, histone deacetylase (HDAC) inhibitors, proteasome inhibitors, cyclooxygenase-2 inhibitors and a number of antibodies which target the DR. Similarly, DNA damaging chemotherapeutic agents targeting Fas (DR2) expression include cisplatin, mitomycin, methotrexate, mitoxantrone, doxorubicin, and bleomycin. Moreover, etoposide, Ara-C, and camptosar (CPT-11) are used to target TRIAL-R1 (DR4) and TRIAL-R2 (DR5), thereby stimulating their expression.80 cFLIP (27 kDa protein) comprises two DEDs which can inhibit FADD and recruited procaspase-8 interaction by binding to DED of FADD and consequently results in the inactivation of caspase-8. A variety of drugs have been developed to trigger the activation of caspases. Such as apoptin (caspase inducing agent) have been utilized for the activation of caspases-3 and caspase-7. Apoptosis facilitates the execution of apoptosis by causing DNA damage and also aid in the release of cytochrome c from mitochondria.84 Moreover, targeting caspase-8 can result in therapeutic effect by utilizing decitabine (5-aza-2´deoxycytidine) which is a cytosine nucleoside analog and endorse demethylation by constraining DNA methyltransferase covalent binding especially in tumors suffering from hypermethylation of caspase-8 promotors, thus restoring the expression of caspase-8.85 Also, gene therapy to induce caspase based apoptosis has adopted by utilizing the genes that encode for inducer, mediator or executioner of apoptosis and also through suppressing the anti-apoptotic gene expression. Selective gene delivery, particular gene expression, and genetic modification by secreting target proteins are some of the strategies adopted to date in apoptosis-based cancer gene therapy.86 The three isoforms of cFLIP in humans include c-FLIPL (long), c-FLIPS (short), and c-FLIPR (splice).60 Generally, higher concentration or enhanced expression of c-FLIPS and c-FLIPL results in the anti-apoptotic function of cFLIP.87 Increased expression of cFLIP observed in different types of cancer leading to enhanced cancer progression.88 Similarly, the reduced expression or down-regulation of cFLIP can inhibit the proliferation of cancer cells and aid in the induction of apoptosis mediated by the DRs and the intrinsic mitochondrial pathway.89 Thus, cFLIP can play an essential role in cancer therapy, specifically if used with TRAIL or conventional chemotherapy.63 Moreover, even though c-FLIPL can perform a dual role in apoptosis (pro- and anti-apoptotic), typically the primary function of cFLIPL have been recognized as an anti-apoptotic regulator of apoptosis in cancer.90cFLIP (cellular FLICE-like) inhibitory protein is a crucial anti-apoptotic regulator that suppresses cell death induced by the DRs such as Fas-L, TNF-α and TRAIL.91 Specifically, siRNA inhibits the expression of c-FLIP and prepare cancer cells to be receptive or sensitize for TRAIL, FASL, and chemotherapeutic agents that induce apoptosis. However, the use of siRNA in vivo involves some restrictions. Besides, the employment of siRNA to inhibit cFLIP depends on the safe delivery of siRNA.92 Many small molecules have been utilized to reduce mRNA and protein intensities of c-FLIPL, however, due to the significantly ordinary homology of cFLIP and caspase-8, use of small molecules for the inhibition of its activity is very challenging.88 Other than utilizing small molecules, different classes of agents have recognized that down-regulate c-FLIP expression by affecting cFLIP transcription, translation and degrading cFLIP.93 These agents include conventional chemotherapeutic drugs, DNA damaging agents and HDAC inhibitors. The conventional chemotherapeutic drugs and DNA damaging agents are cisplatin, doxorubicin, camptothecin, 9-nitrocamptothecin, and oxaliplatin. HDAC inhibitors include SAHA and the inhibitors of MEK1/2, PKC, and PI3K.86 DR agonists represent an effective therapeutics that mainly target apoptosis. Further, clinical trials of these agents showed the safety of the approach and apoptotic cell death. Forthcoming data from recent trials will also help to demonstrate their clinical activity in different tumor types alone and combinations. Although understanding the mechanism of the TRAIL pathway, studying various factors that might halt response, win over the mechanisms of tumor-cell resistance, and get benefit from these therapies.Various approaches that have taken to reduce or suppress the anti-apoptotic function of cFLIP involve the use of siRNA , use of many small molecules and agents that down-regulate cFLIP.94 The categorization of Bcl-2 family proteins based on the existence of shared blocks of sequence homology, named as Bcl-2 homology (BH). The equilibrium between pro- and anti-apoptotic Bcl-2 family proteins is an essential element for the initiation of mitochondrial outer membrane permeabilization (MOMP).95 Bcl-2, Bcl-XL, and Mcl-1 are anti-apoptotic proteins and their role is to prevent the release of cytochrome c and maintain mitochondrial integrity while Bax, Bak, Bad, and Bok are pro-apoptotic proteins of Bcl-2 family which allow the release of cytochrome c from the mitochondrial intermembrane space into the cytosol to promote the induction of apoptosis eventually aid in cancer therapeutics.52 Up-regulation of pro-apoptotic Bcl-2 proteins and down-regulation of anti-apoptotic Bcl-2 effectively linked to the mechanism of cell death. For example, the ratio between pr-apoptotic (Bax) and anti-apoptotic (Bcl-2) proteins is generally used to determine the fate of the cell.96 Inactivation of pro-apoptotic proteins with multidomain (Bax and Bak) is a crucial feature of carcinogenesis.97 Similarly, elevated levels of anti-apoptotic proteins multidomain  encourage the deregulation of apoptosis in cancer cells and also aid cancer cells to become resistant to immune-surveillance.98 However, single proapoptotic domain BH3, i.e., BID, BIM, BAD, PUMA (p53 upregulated controller of apoptosis) and NOXA play a primary role in regulating and triggering apoptosis act as sensitizer and serve as an excellent therapeutic target. The overexpression of anti-apoptotic Bcl-2 family members or underexpression of pro-apoptotic Bcl-2 family members usually associated with chemoresistance. Further, BCL-2 over-expression has found in acute myeloid leukemia, chronic lymphocytic leukemia (CLL), non-Hodgkin’s lymphoma (NHL), myeloma, melanoma and hepatocellular, lung, breast, prostate carcinomas.101Bcl-2 family proteins that comprise of pro- and anti-apoptotic proteins are known to play an essential role in the regulation of intrinsic pathway of apoptosis.102 Effective strategies have been adopted to inhibit the anti-apoptotic effects of Bcl-2 family, which include: using antisense oligonucleotides, development of small drug molecules and inhibit the gene transcription.96 An example of novel Bcl-2 antisense is Oblimersen Sodium , 18-base antisense phosphorothioate oligonucleotide used in I and II phase of clinical trials in advance solid cancer lymphoma.104 It has also tested in combination with other anticancer agents, such as Oblimersen with rituximab used for NHL in II phase, Oblimersen with dacarbazine for myeloma in III phase, Oblimersen with docetaxel for castration-resistant prostate cancer (CRPC) and breast cancer in II and I phase, non-small-cell lung carcinoma (NSCLC) or small-cell lung carcinoma (SCLC) in III phase and HRPCa (EORTC) in II phase.109Potential therapeutic agents with improved efficacy have been developed to target the down-regulation of anti-apoptotic and up-regulating pro-apoptotic Bcl-2 protein.110 A significant controllers of gene expression work enzymatically in removing the acetyl group from histones proteins.113 Genetic knock-down has been shown the role of HDACs induce apoptosis and cell cycle arrest in different tumor types, such as colon, lung, breast carcinomas and acute promyelocytic leukemia, highlighting its activity as a critical indicator of survival in cancer cells.114 Further, over-expression of HDACs has been linked to various critical events of tumorigenesis, includes epigenetic repression of CDKN1A (encoding the cyclin-dependent kinase inhibitor p21) tumor suppressor gene and essential genes, like breast cancer 1, early onset BRCA1 and ataxia telangiectasia and Rad 3 related (ATR).95 The Sodium butyrate is small molecule Inhibitors (HDAC inhibitor) involved in gene expression alteration in regulation of proapoptotic proteins. Another molecule Flavopiridol (cyclin-dependent kinase inhibitor) and are used to down-regulate the Bcl-2 and Bcl-xl, Mcl-1 expression respectively. Also, Fenretinide, which is a synthetic cytotoxic retinoid, acts by down-regulating the activity of Bcl-2 and Mcl-1 without altering the expression of pro-apoptotic protein Bax.40 Many HDACi have entered phase I to III clinical trials such as, CHR-3996 used for a Refractory solid tumor in phase I.115 Another inhibitory agent Panobinostat (LBH589) used for Relapsed or refractory NHL and advanced solid tumors and Panobinostat (LBH589) along with melphalan for Relapsed or multiple refractory myelomas in I and II phase.118HDACs are attractive therapeutic targets in cancer and inflammatory diseases.122 All BH3 proteins composed of the single domain called α-helical BH3 domain has been demonstrated to play a crucial role in cancer therapy.125 The BH-3 mimetics have developed which precisely bind to the hydrophobic groove (which facilitate the binding between pro- and anti-apoptotic proteins), and by this means they oppose the function of anti-apoptotic Bcl-2 family proteins.95 Bcl-2 antisense causes the down-regulation of Bcl-2 proteins by affecting the corresponding mRNA. Potential BH-3 mimetic drugs such as, Obatoclax Mesylate (GX15- 070MS) for SCLC and myelofibrosis and Gossypol/ AT-101 for Metastatic breast cancer and CRPC in I & II phase which inhibits Bcl-2, Bcl-xL, and Mcl- 1 expression. ATB-263 and ATB-199 inhibit/block Bcl-2 for Advanced hematological cancers and CLL 73,74 and MIM1 which inhibits Mcl-1 in clinical trials.126Natural and synthetic small-molecules BH3-mimicking agents have successfully antagonized antiapoptotic Bcl-2 protein family members, such as Obatoclax, Gossypol, ATB-263 and ATB-199.127 Tumor suppressor genes are responsible for controlling DNA repair and cell division. dysfunctional tumor suppressor genes could result in uncontrolled multiplication of cells leading to cancer. Many aspects like chemicals, ionizing radiation, and viruses can cause alterations in proto-oncogenes and tumor suppressor genes.129 The expression of p53 is deficient in normal cells under non-stressed conditions. However, p53 can be activated by any stress stimuli; DNA damage or in the response of oncogene activation. Extra and intracellular stress signals change latent p53 to an active form and encourage p53 to accumulate in a cell nucleus. The stability of activated p53 regulated through various post-translational chemical modifications like phosphorylation, acetylation, and methylation.131 The fundamental role of p53 is its capability to induce apoptosis by transcription-dependent and transcription-independent manner. p53 performs its function by transcription activation of pro-apoptotic Bcl-2 family proteins and transcription suppression of anti-apoptotic Bcl-2 family proteins. Moreover, it can directly interact with Bax that successively stimulates the release of cyt C via MOMP and aid in the induction of apoptosis.132Tumor suppressor gene p53 is the primary entities involved in carcinogenesis plays an essential part in cancer concerning both cell cycle arrest, and apoptosis.135 However, the pharmacological action of Nutlin-3 is via both the transcription-dependent and - independent p53 apoptotic pathways.137 Nutlin-3 has been shown to induce mitotic arrest rather than apoptosis mainly.138 MDM2 can also trigger, in response to low genotoxic damage, the downregulation of p53 apoptotic activator HIPK2.139 Interestingly, Nutlin-3 along with zinc ion inhibit the MDM2 ligase activity favoring HIPK2 stabilization results in an induction of p53 apoptotic activity.140 Similarly, co-treatment of Nutlin-3 and (ABT-737) Bcl-2 inhibitor has been shown to enhance the sensitivity to apoptosis of cancer cells greatly.141 Further, multi-target anticancer approach, inhibition of both MDM2 and Bcl-2 could be a positive tool in cancer treatment.142 Another reported small molecule MDM2 inhibitor is CP-31398, which increases the transcriptional activity of p53 in cells.143 Furthermore, three different types of p53 vaccines such as peptide-based vaccines, dendritic-cell based vaccines, and recombinant virus-based vaccines are undergoing clinical trials to assist in the induction of antitumor immune responses.146 Another research reveals a class of small molecules that reactivates the wild-type function of mutant p53 in so doing permit p53 to induce apoptotic cell death. PRIMA-1 and its analog APR-246 are examples of this class of small molecules and are undergoing preclinical and clinical trials (phase I) to functions as reactivating mutant p53.147Different small molecules MDM2 inhibitors that have been developed to trigger wild-type p53 activity, such as Nultlin-3, MI-219, and RITA. The role of Nultlin-3 and MI-219 is to prevent the interaction of MDM2 and p53, activating p53 signaling and suppressing the tumor growth.148 IAPs can inhibit both intrinsic and extrinsic pathway of apoptosis. The execution of DR-mediated extrinsic pathway and mitochondrial triggered a family of structurally diverse IAPs modulates intrinsic pathway; X-linked (XIAP), cellular , neuronal (NIAP), testis-specific (Ts-IAP), Bir-ubiquitin conjugating enzyme (BRUCE), Survivin and Livin. Structurally, IAPs are approximately 70 amino acids long and contain zinc finger BIR (Baculovirus IAP Repeat) domains that are responsible for deregulation properties of IAPs where they prevent the conversion of zymogenic (inactive) pro-caspases to active caspases.150 Over-expression of IAPs linked to increased chemo-resistance in several types of cancer.152 As a controlled expression of IAPs could encourage apoptotic cell death, different strategies have been adopted to inhibit IAPs, and these include: anti-sense facilitated interference of XIAP and survivin oligonucleotides and siRNA expression and inhibition of IAPs by SMAC mimetic compounds.155 XIAP inhibits both the extrinsic and intrinsic apoptotic pathways via direct inhibition of enzymes caspases and may be limited by its initiation of cell protective effects via NF-kB signaling and cIAP1/2 through proteasomal degradation or ubiquitination.156 Knockout strategies in cancer cells were highlighting their role in resistance to various anti-cancer therapies. For example, increased apoptosis suppressed tumorigenicity and re-sensitized was reported ovarian cancer cells to cisplatin therapy and in nude mice through shRNA mediated knockdown of XIAP.157 The successful results in acute myeloid leukemia patients undergoing therapy using antisense oligonucleotide AEG35156 that target XIAP in phase II trials.159 However, despite this initial success and confirmed on-target knockdown,157 a later trial failed to report a similarly improved outcome in patients with advanced pancreatic cancer.160 while gene silencing is an attractive prospect, its potential clinical relevance is limited by lower knockdown efficiency in patient samples, compared to those demonstrated in cell culture.158 and by the transient nature of XIAP repression.160 Still, strategies for RNAi remain important tools to dissect the mechanistic and functional role of IAPs in cancer.The IAPs is family of protein which functions as endogenous inhibitors of apoptosis. Elevated expression levels of IAPs were significantly resulting in improved cell survival, increased tumor growth and consequent metastasis. IAPs targeting strategy has become increasingly attractive to sensitize cancer cells towards various therapeutics such as chemotherapies, antibody based-therapies. Besides apoptosis, IAPs observed to play a part in necroptosis, immune regulation, chromosomal and cytoplasmic division.161 To date, some inhibitors of IAP proteins have been developed these include: SH122, SH130, SM164, AZD5582, JP1201, AEG35156, LY2181308 and YM155.151 SMAC mimetic SH130 and SH122 target human prostate cancer cell line by inhibiting IAPs.163 AZD5582 and JP1201 are SMAC mimetics which target CLL and pancreatic cancer cell line respectively to enhance apoptosis by TRAIL.165 YM155 suppresses survivin expression and induce apoptosis in human cancer cell lines.167 AEG35156 and LY2181308 are antisense oligonucleotides and small siRNA molecules which targets survivin expression and also down-regulate XIAP.169 Various past researches demonstrated that SMAC mimetics, along with anticancer drugs and TRAIL remarkably enhance apoptotic cell death in several cancer cell types in vitro, such as T98G glioblastoma cells, HeLa cells, and lung adenocarcinomas.172 Further, the SMAC mimetics role as sensitizer enhance the sensitivity of various agents, such as paclitaxel, etoposide, and doxorubicin in MCF-7 breast cancer cells.173 Various apoptotic pathways targeted strategies as shown in SMAC mimetics release into the cytosol as a result of MOMP binds to the BIR domain cellular IAPs (cIAP1 and cIAP2) and XIAP, restoring the function of effector caspases by blocking inhibitory role of IAPs.Targeting apoptosis is a new standard in cancer drug development: a significant regulatory mechanism inside cells, a cellular process that is tightly regulated by various membrane-bound and freely available cytoskeleton molecules. Apoptosis is a fundamental regulatory mechanism of normal cells, any dysregulation in apoptosis could trigger the uncontrol multiplication of cells. This regulatory process characterized by a series of specific morphological changes along with biochemical features which involve extrinsic and intrinsic pathways via a different protein that plays a crucial role overall. Therefore, a detailed mechanistic understanding of the apoptotic signaling pathways required for the development of effective cancer therapeutics. The up-regulation of apoptotic pathways via activation of pro-apoptotic pathway proteins . The understanding of apoptotic pathways needs intense effort for the development of new approaches to drug discovery and therapy. However, some apoptotic pathways proteins which induce apoptosis selected as a target for drugs are in clinical trials. Positive results of antibodies along with recombinant TRAIL specifically target the DRs in clinical trials against a range of solid tumors. However, much understanding of evading apoptosis in cancer cells is needed to get the positive results in maturing clinical data. The novel agents along with combined apoptotic inhibitors strategy show significant synergistic effects and being in the current study.Not applicable.The authors declare no conflict of interest.