Unwinding Helicase MCM Functionality for Diagnosis and Therapeutics of Replication Abnormalities Associated with Cancer: A Review.

The minichromosome maintenance (MCM) protein is a component of an active helicase that is essential for the initiation of DNA replication. Dysregulation of MCM functions contribute to abnormal cell proliferation and genomic instability. The interactions of MCM with cellular factors, including Cdc45 and GINS, determine the formation of active helicase and functioning of helicase. The functioning of MCM determines the fate of DNA replication and, thus, genomic integrity. This complex is upregulated in precancerous cells and can act as an important tool for diagnostic applications. The MCM protein complex can be an important broad-spectrum therapeutic target in various cancers. Investigations have supported the potential and applications of MCM in cancer diagnosis and its therapeutics. In this article, we discuss the physiological roles of MCM and its associated factors in DNA replication and cancer pathogenesis.

Deciphering the molecular functionality of Cdc45 in replisomal complex.

The members of DHH superfamily have been reported with diverse substrate spectrum and play pivotal roles in replication, repair, and RNA metabolism. This family comprises phosphatases, phosphoesterase and bifunctional enzymes having nanoRNase and phosphatase activities. Cell cycle factor Cdc45, a member of this superfamily, is crucial for movement of the replication fork during DNA replication and an important component of the replisome. The specific protein-protein interactions of Cdc45 with other factors along with helicase moderate the faithful DNA replication process. However, the exact biochemical functions of this factor are still unknown and need further investigation. Here, we studied the biochemical roles of Cdc45 and its molecular interactions within the replisomal complex. The alteration in the level of protein, observed when DNA damage is induced in-vivo, suggests its association with DNA replication stress. We analyzed protein Cdc45, providing new insights about the molecular and biochemical functionality of this replisomal factor.

A broadly neutralizing monoclonal antibody overcomes the mutational landscape of emerging SARS-CoV-2 variants of concern.

The emergence of new variants of SARS-CoV-2 necessitates unremitting efforts to discover novel therapeutic monoclonal antibodies (mAbs). Here, we report an extremely potent mAb named P4A2 that can neutralize all the circulating variants of concern (VOCs) with high efficiency, including the highly transmissible Omicron. The crystal structure of the P4A2 Fab:RBD complex revealed that the residues of the RBD that interact with P4A2 are a part of the ACE2-receptor-binding motif and are not mutated in any of the VOCs. The pan coronavirus pseudotyped neutralization assay confirmed that the P4A2 mAb is specific for SARS-CoV-2 and its VOCs. Passive administration of P4A2 to K18-hACE2 transgenic mice conferred protection, both prophylactically and therapeutically, against challenge with VOCs. Overall, our data shows that, the P4A2 mAb has immense therapeutic potential to neutralize the current circulating VOCs. Due to the overlap between the P4A2 epitope and ACE2 binding site on spike-RBD, P4A2 may also be highly effective against a number of future variants.

Characterization of a broadly cross reactive tetravalent human monoclonal antibody, recognizing conformational epitopes in receptor binding domain of SARS-CoV-2.

We used human semi-synthetic phage antibody gene libraries to select anti-SARS-CoV-2 RBD scFv antibody fragment and subsequent characterization of this novel tetravalent monoclonal antibody targeting conformational epitopes in the receptor binding domain of SARS-CoV-2. Binding studies suggest that II62 tetravalent antibody cross-reacts with RBD protein of SARS-CoV2 and its different variants of concerns. The epitope mapping data reveals that II62 tetravalent antibody targets an epitope that does not directly interferes with RBD: ACE2 interaction. Neutralization studies with live authentic SARS-CoV2 virus suggests that increase in valency of II62 mAb from monovalent to tetravalent doesn't perturbate virus interactions with the ACE2 expressing host cells in cytopathic effect-based (CPE) assay. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03272-6.

Protocol for High Throughput Screening of Antibody Phage Libraries.

Phage display is a proven and widely used technology for selecting specific antibodies against desired targets. However, an immense amount of effort is required to identify and screen the desired positive clones from large and diverse combinatorial libraries. On the other hand, the selection of positive binding clones from synthetic and semi-synthetic libraries has an inherent bias toward clones with randomly produced amber stop codons, making it more difficult to identify desirable binding antibodies. To overcome the screening of desired clones with amber codons, we present a step-by-step approach for effective phage library screening to isolate useful antibodies. The procedure calls for creating a simple new vector system for soluble production of phage ELISA positive binding clones with one or more amber stop codons in their single-chain antibody fragment (scFv) gene sequences, which is otherwise difficult in standard screening. Graphical abstract.

Investigating a putative transcriptional regulatory protein encoded by Rv1719 gene of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis, demonstrates immense plasticity with which it adapts to a highly dynamic and hostile host environment. This is facilitated by a web of signalling pathways constantly modulated by a multitude of proteins that regulate the flow of genetic information inside the pathogen. Transcription factors (TFs) belongs to one such family of proteins that modulate the signalling by regulating the abundance of proteins at the transcript level. In the current study, we have characterized the putative transcriptional regulatory protein encoded by the Rv1719 gene of Mycobacterium tuberculosis. This TF belongs to the IclR family of proteins with orthologs found in both bacterial and archaeal species. We cloned the Rv1719 gene into the pET28a expression vector and performed heterologous expression of the recombinant protein with E coli as the host. Further, optimization of the purification protocol by affinity chromatography and characterization of proteins for their functional viability has been demonstrated using various biochemical and/or biophysical approaches. Scale-up of purification yielded approximately 30 mg of ~ 28 kDa protein per litre of culture. In-silico protein domain analysis of Rv1719 protein predicted the presence of the helix-turn-helix (HTH) domain suggesting its ability to bind DNA sequence and modulate transcription; a hallmark of a transcriptional regulatory protein. Further, by performing electrophoretic mobility shift assay (EMSA) we demonstrated that the protein binds to a specific DNA fragment harboring the probable binding site of one of the predicted promoters.

The SARS CoV-2 spike directed non-neutralizing polyclonal antibodies cross-react with Human immunodeficiency virus (HIV-1) gp41.

Cross-reactivity among the two diverse viruses is believed to originate from the concept of antibodies recognizing similar epitopes on the two viral surfaces. Cross-reactive antibody responses have been seen in previous variants of SARS and SARS-CoV-2, but little is known about the cross reactivity with other similar RNA viruses like HIV-1. In the present study, we examined the reactivity the SARS-CoV-2 directed antibodies, via spike, immunized mice sera and demonstrated whether they conferred any cross-reactive neutralization against HIV-1. Our findings show that SARS-CoV-2 spike immunized mice antibodies cross-react with the HIV-1 Env protein. Cross-neutralization among the two viruses is uncommon, suggesting the presence of a non-neutralizing antibody response to conserved epitopes amongst the two viruses. Our results indicate, that SARS-CoV-2 spike antibody cross reactivity is targeted towards the gp41 region of the HIV-1 Env (gp160) protein. Overall, our investigation not only answers a crucial question about the understanding of cross-reactive epitopes of antibodies generated in different viral infections, but also provides critical evidence for developing vaccine immunogens and novel treatment strategies with enhanced efficacy capable of recognising diverse pathogens with similar antigenic features.

Inhalation monoclonal antibody therapy: a new way to treat and manage respiratory infections.

The route of administration of a therapeutic agent has a substantial impact on its success. Therapeutic antibodies are usually administered systemically, either directly by intravenous route, or indirectly by intramuscular or subcutaneous injection. However, treatment of diseases contained within a specific tissue necessitates a better alternate route of administration for targeting localised infections. Inhalation is a promising non-invasive strategy for antibody delivery to treat respiratory maladies because it provides higher concentrations of antibody in the respiratory airways overcoming the constraints of entry through systemic circulation and uncertainity in the amount reaching the target tissue. The nasal drug delivery route is one of the extensively researched modes of administration, and nasal sprays for molecular drugs are deemed successful and are presently commercially marketed. This review highlights the current state and future prospects of inhaled therapies, with an emphasis on the use of monoclonal antibodies for the treatment of respiratory infections, as well as an overview of their importance, practical challenges, and clinical trial outcomes.Key points• Immunologic strategies for preventing mucosal transmission of respiratory pathogens.• Mucosal-mediated immunoprophylaxis could play a major role in COVID-19 prevention.• Applications of monoclonal antibodies in passive immunisation.

Non-neutralizing SARS CoV-2 directed polyclonal antibodies demonstrate cross-reactivity with the HA glycans of influenza virus.

The spike protein of the SARS-CoV-2 virus is the foremost target for the designing of vaccines and therapeutic antibodies and also acts as a crucial antigen in the assessment of COVID-19 immune responses. The enveloped viruses; such as SARS-CoV-2, Human Immunodeficiency Virus-1 (HIV-1) and influenza, often hijack host-cell glycosylation pathways and influence pathobiology and immune selection. These glycan motifs can lead to either immune evasion or viral neutralization by the production of cross-reactive antibodies that can lead to antibody-dependent enhancement (ADE) of infection. Potential cross-protection from influenza vaccine has also been reported in COVID-19 infected individuals in several epidemiological studies recently; however, the scientific basis for these observations remains elusive. Herein, we show that the anti-SARS-CoV2 antibodies cross-reacts with the Hemagglutinin (HA) protein. This phenomenon is common to both the sera from convalescent SARS-CoV-2 donors and spike immunized mice, although these antibodies were unable to cross-neutralize, suggesting the presence of a non-neutralizing antibody response. Epitope mapping suggests that the cross-reactive antibodies are targeted towards glycan epitopes of the SARS-CoV-2 spike and HA. Overall, our findings address the cross-reactive responses, although non-neutralizing, elicited against RNA viruses and warrant further studies to investigate whether such non-neutralizing antibody responses can contribute to effector functions such as antibody-dependent cellular cytotoxicity (ADCC) or ADE.

A review on genotoxicity in connection to infertility and cancer.

Genotoxicity has been identified as the main cause of infertility and a variety of cancers. The mechanisms affect the structure, quality of the information or the segregation of DNA and are not inherently correlated with mutagenicity. The concept of genotoxicity, the chemical classes that cause genetic damage and the associated mechanisms of action are discussed here. Hazardous effects of pharmaceuticals, cosmetics, agrochemicals, industrial compounds, food additives, natural toxins and nanomaterials are, in large part, identified by genotoxicity and mutagenicity tests. These are critical and early steps in industrial and regulatory health assessment. Though several in vitro experiments are commonly used and approval by regulatory agencies for commercial licensing of drugs, their accuracy in human predictions for genotoxic and mutagenic effects is frequently questioned. Treatment of real and functional genetic toxicity problems depends in detail on the knowledge of mechanisms of DNA damage in the molecular, subcellular, cellular and tissue or organ system levels. Current strategies for risk assessment of human health need revisions to achieve robust and reliable results for optimizing their effectiveness. Additionally, computerized methods, neo-biomarkers leveraging '-omics' approaches, all of which can provide a convincing genotoxicity evaluation to reduce infertility and cancer risk.

A rapid novel strategy for screening of antibody phage libraries for production, purification, and functional characterization of amber stop codons containing single-chain antibody fragments.

Phage display antibody (PDA) libraries, allows the rapid isolation and characterization of high specificity monoclonal antibodies for therapeutic and diagnostic applications. However, selection of positive binding clones from synthetic and semi-synthetic libraries has an inherent bias towards clones containing randomly generated amber stop codons, complicating the identification of high affinity binding antibodies. We screened Tomlinson I and J library against receptor binding domain (RBD) of SARS CoV2, eight clones which showed positive binding in phage ELISA, contained one or more amber stop codons in their single-chain antibody fragment (scFv) gene sequences. The presence of amber stop codons within the antibody sequence causes the premature termination of soluble form of scFv expression in nonsuppressor Escherichia coli strain. In the present study, we have used a novel strategy that allows soluble expression of scFvs having amber stop codon in their gene sequences (without phage PIII protein fusion), in the suppressor strain. This strategy of introduction of Ochre (TAA) codon at the junction of scFv and PIII gene, speeds up the initial screening process which is critical for selecting the right scFvs for further studies. Present strategy leads to the identification of a scFv, B8 that binds specifically with nanomolar affinity toward SARS CoV 2 RBD, which otherwise lost in terms of traditional methodology.

Tmprss2 specific miRNAs as promising regulators for SARS-CoV-2 entry checkpoint.

Tmprss2 is an emerging molecular target which guides cellular infections of SARS-CoV-2, has been earmarked for interventions against the viral pathologies. The study aims to computationally screen and identifies potential miRNAs, following in vitro experimental validation of miRNA-mediated suppression of Tmprss2 for early prevention of COVID-19. Pool of 163 miRNAs, scrutinized for Tmprss2 binding with three miRNA prediction algorithms, ensued 11 common miRNAs. Further, computational negative energies for association, corroborated miRNA-Tmprss2 interactions, whereas three miRNAs (hsa-miR-214, hsa-miR-98 and hsa-miR-32) based on probability scores ≥0.8 and accessibility to Tmprss2 target have been selected in the Sfold tool. Transfection of miRNA(s) in the Caco-2 cells, quantitatively estimated differential expression, confirming silencing of Tmprss2 with maximum gene suppression by hsa-miR-32 employing novel promising role in CoV-2 pathogenesis. The exalted binding of miRNAs to Tmprss2 and suppression of later advocates their utility as molecular tools for prevention of SARS-CoV-2 viral transmission and replication in humans.

miR300 intervenes Smad3/ß-catenin/RunX2 crosstalk for therapy with an alternate function as indicative biomarker in osteoporosis.

The study reports a theranostic nature of rno-miR-300 (miR300) in the osteoblast functioning, by influencing the signaling pathway(s), associated with osteoblast differentiation. Excessive expression of miR300 suppresses osteoblast functions. Smad3 served as a validated target for miR300, on homology-based computational analysis and experimental testimony, which activates ß-catenin, and subsequently potentiates Runx2. The impact of miR300 on the Smad3/ß-catenin/Runx2 signaling interactions in the induction of osteoblast differentiation was scrutinized by immunoblotting and in vivo miRNA antagonism. Overexpression of miR300 in the rat calvarial osteoblasts decreases the protein levels of Smad3, ß-catenin and Runx2. Besides, in vivo silencing of miR300 in the neonatal pups and adult rats by AntimiR300 abolishes the suppressing action of miR300 on the osteoblast differentiation and expressions of Smad3/ß-catenin/Runx2 axis. MicroCT studies showed improved trabecular microarchitecture in the AntimiR300 transfected ovariectomised rat model compared to sham and negative control. Furthermore, expression levels of miR300 were evaluated in serum samples from an independent set of 30 osteoporotic patients followed by a Receiver Operating Characteristic Curve (ROC) based analysis for the diagnostic efficiency of miR300. Interestingly, the results exhibited high levels of miR300 (p < 0.0001) in the serum samples from osteoporotic patients relative to non-osteoporotic subjects (AUC = 0.9689). Thus, miR300 negatively regulates the differentiation of osteoblasts by targeting crosstalk among Smad3, ß-catenin and Runx2, unveiling an enormous ability to serve as a therapeutic target for bone-related disorder management strategies. Besides, miR300 may potentially function for the diagnosis of osteoporosis as a non-invasive biomarker.

Role of VapBC12 Toxin-Antitoxin Locus in Cholesterol-Induced Mycobacterial Persistence.

The worldwide increase in the frequency of multidrug-resistant and extensively drug-resistant cases of tuberculosis is mainly due to therapeutic noncompliance associated with a lengthy treatment regimen. Depending on the drug susceptibility profile, the treatment duration can extend from 6 months to 2 years. This protracted regimen is attributed to a supposedly nonreplicating and metabolically inert subset of the Mycobacterium tuberculosis population, called "persisters." The mechanism underlying stochastic generation and enrichment of persisters is not fully known. We have previously reported that the utilization of host cholesterol is essential for mycobacterial persistence. In this study, we have demonstrated that cholesterol-induced activation of a RNase toxin (VapC12) inhibits translation by targeting proT tRNA in M. tuberculosis This results in cholesterol-specific growth modulation that increases the frequency of generation of the persisters in a heterogeneous M. tuberculosis population. Also, a null mutant strain of this toxin (ΔvapC12) demonstrated an enhanced growth phenotype in a guinea pig model of M. tuberculosis infection, depicting its role in disease persistence. Thus, we have identified a novel strategy through which cholesterol-specific activation of a toxin-antitoxin module in M. tuberculosis enhances persister formation during infection. The current findings provide an opportunity to target persisters, a new paradigm facilitating tuberculosis drug development.IMPORTANCE The current TB treatment regimen involves a combination of drugs administered for an extended duration that could last for 6 months to 2 years. This could lead to noncompliance and the emergence of newer drug resistance strains. It is widely perceived that the major culprits are the so-called nonreplicating and metabolically inactive "persister" bacteria. The importance of cholesterol utilization during the persistence stage of M. tuberculosis infection and its potential role in the generation of persisters is very intriguing. We explored the mechanism involved in the cholesterol-mediated generation of persisters in mycobacteria. In this study, we have identified a toxin-antitoxin (TA) system essential for the generation of persisters during M. tuberculosis infection. This study verified that M. tuberculosis strain devoid of the VapBC12 TA system failed to persist and showed a hypervirulent phenotype in a guinea pig infection model. Our studies indicate that the M. tuberculosis VapBC12 TA system acts as a molecular switch regulating persister generation during infection. VapBC12 TA system as a drug target offers opportunities to develop shorter and more effective treatment regimens against tuberculosis.

Identification of an anti-SARS-CoV-2 receptor-binding domain-directed human monoclonal antibody from a naïve semisynthetic library.

There is a desperate need for safe and effective vaccines, therapies, and diagnostics for SARS- coronavirus 2 (CoV-2), the development of which will be aided by the discovery of potent and selective antibodies against relevant viral epitopes. Human phage display technology has revolutionized the process of identifying and optimizing antibodies, providing facile entry points for further applications. Herein, we use this technology to search for antibodies targeting the receptor-binding domain (RBD) of CoV-2. Specifically, we screened a naïve human semisynthetic phage library against RBD, leading to the identification of a high-affinity single-chain fragment variable region (scFv). The scFv was further engineered into two other antibody formats (scFv-Fc and IgG1). All three antibody formats showed high binding specificity to CoV-2 RBD and the spike antigens in different assay systems. Flow cytometry analysis demonstrated specific binding of the IgG1 format to cells expressing membrane-bound CoV-2 spike protein. Docking studies revealed that the scFv recognizes an epitope that partially overlaps with angiotensin-converting enzyme 2 (ACE2)-interacting sites on the CoV-2 RBD. Given its high specificity and affinity, we anticipate that these anti-CoV-2 antibodies will be useful as valuable reagents for accessing the antigenicity of vaccine candidates, as well as developing antibody-based therapeutics and diagnostics for CoV-2.

Hybridoma technology a versatile method for isolation of monoclonal antibodies, its applicability across species, limitations, advancement and future perspectives.

The advancements in technology and manufacturing processes have allowed the development of new derivatives, biosimilar or advanced improved versions for approved antibodies each year for treatment regimen. There are more than 700 antibody-based molecules that are in different stages of phase I/II/ III clinical trials targeting new unique targets. To date, approximately more than 80 monoclonal antibodies (mAbs) have been approved. A total of 7 novel antibody therapeutics had been granted the first approval either in the United States or European Union in the year 2019, representing approximately 20% of the total number of approved drugs. Most of these licenced mAbs or their derivatives are either of hybridoma origin or their improvised engineered versions. Even with the recent development of high throughput mAb generation technologies, hybridoma is the most favoured method due to its indigenous nature to preserve natural cognate antibody pairing information and preserves innate functions of immune cells. The recent advent of antibody engineering technology has superseded the species level barriers and has shown success in isolation of hybridoma across phylogenetically distinct species. This has led to the isolation of monoclonal antibodies against human targets that are conserved and non-immunogenic in the rodent. In this review, we have discussed in detail about hybridoma technology, its expansion towards different animal species, the importance of antibodies isolated from different animal sources that are useful in biological applications, advantages, and limitations. This review also summarizes the challenges and recent progress associated with hybridoma development, and how it has been overcome in these years to provide new insights for the isolation of mAbs.

Expression, Purification and Characterization of the Hepatitis E Virus Like-Particles in the Pichia pastoris.

Hepatitis E virus (HEV) is associated with acute hepatitis disease, which may lead to chronic disease in immunocompromised individuals. The disease is particularly severe among pregnant women (20-30% mortality). The only licensed vaccine against HEV, which is available in China, is the Escherichia coli purified recombinant virus-like particles (VLPs) encompassing the 368-660 amino acids (aa) of the viral ORF2 protein. The viral capsid is formed by the ORF2 protein, which harbors three glycosylation sites. Baculo virus expression system has been employed to generate a glycosylated VLP, which encompasses 112-608aa of the ORF2 protein. Here, we sought to produce a recombinant VLP containing 112-608aa of the ORF2 protein in Pichia pastoris (P. pastoris) expression system. The cDNA sequence encoding 112-608aa of the ORF2 protein was fused with the α-mating factor secretion signal coding sequence (for release of the fusion protein to the culture medium) and cloned into the yeast vector pPICZα. Optimum expression of recombinant protein was obtained at 72 h induction in 1.5% methanol using inoculum density (A600) of 80 and at pH-3.0 of the culture medium. Identity of the purified protein was confirmed by mass spectrometry analysis. Further studies revealed the glycosylation pattern and VLP nature of the purified protein. Immunization of BALB/c mice with these VLPs induced potent immune response as evidenced by the high ORF2 specific IgG titer and augmented splenocyte proliferation in a dose dependent manner. 112-608aa ORF2 VLPs produced in P. pastoris appears to be a suitable candidate for development of diagnostic and prophylactic reagents against the hepatitis E.

Serodiagnostic evaluation of recombinant CdtB of S. Typhi as a potential candidate for acute typhoid.

Typhoid fever caused by human restricted Salmonella typhi presents a considerable health burden on developing South-Asian nations like India. The suboptimal sensitivity and specificity associated with culture-based isolation of etiological agent and the extensively used surface antigen-based serological assays often lead to misdiagnosis and inappropriate antimicrobial treatment. The increasing reports of the emergence of resistant strains and undefined disease burden signify the critical need for an inexpensive, reliable, easy-to-use, and highly sensitive diagnostic test for typhoid fever. Utilizing S. typhi-specific and immunogenic antigens in sero-diagnostic assays could lead to precise diagnosis of acute typhoid and prompt treatment. In this study, we report cloning, expression, and purification of recombinant Cytolethal distending toxin subunit B (CdtB) of S. typhi, which is reported to be highly specific, immunogenic, and expressed only upon S. typhi infection. We further evaluated the purified recombinant CdtB for its diagnostic potential in an IgM-based indirect ELISA format using 33 human samples. Twenty-one serum samples from blood culture confirmed cases (n = 21) of typhoid and 12 samples from healthy controls (n = 12) were tested. The assay showed sensitivity of 100% and specificity of 83.3% respectively with positive and negative predictive values of 91.3 and 100% respectively. Efficient detection of specific IgM antibodies indicates that CdtB could be highly valuable in sero-diagnosis of acute typhoid and rapid screening of clinical samples.

Scriptaid overcomes hypoxia-induced cisplatin resistance in both wild-type and mutant p53 lung cancer cells.

Non-small cell lung cancer (NSCLC), comprising 85% of lung cancer cases, has been associated with resistance to chemo/radiotherapy. The hypoxic tumor micro-environment, where insufficient vasculature results in poor drug penetrance and sub-optimal chemotherapy in the tumor interiors contributes heavily to this resistance. Additionally, epigenetic changes in tumorigenic cells also change their response to different forms of therapy. In our study, we have investigated the effectiveness of a combination of cisplatin with scriptaid [a pan-Histone Deacetylase inhibitor (HDACi)] in a model that mimics the tumor microenvironment of hypoxia and sub-lethal chemotherapy. Scriptaid synergistically increases the efficacy of cisplatin in normoxia as well as hypoxia, accompanied with reduced metastasis and enhanced DNA damage. Addition of scriptaid also overcomes the cisplatin resistance exhibited in lung cancer cells with stabilized hypoxia inducible factor 1 (HIF1)-α (mutant) and mutant p53. Molecular studies showed that the combination treatment increased apoptotic cell death in both normoxia and hypoxia with a dual role of p38MAPK. Together, our results suggest that the combination of low dose cisplatin and scriptaid is cytotoxic to NSCLC lines, can overcome hypoxia induced resistance and mutant p53- induced instability often associated with this cancer, and has the potential to be an effective therapeutic modality.

Response of primary culture of human ovarian cancer cells to chemotherapy: In vitro individualized therapy.

OBJECTIVE: This study focused on whether primary cultures of ovarian cancer (OC) cells established from ascites can be used to evaluate response to chemotherapeutic agents and if curcumin could enhance the efficacy of these agents. MATERIALS AND METHODS: We established five primary cultures of ascitic cells from OC patients and treated them with curcumin, carboplatin, and paclitaxel singly and in combinations. The percentage of apoptotic cells was determined by flow cytometry. RESULTS: There was a wide variation in the response of individual primary cultures to treatment with the chemotherapeutic agents. Curcumin by itself was as good as carboplatin or paclitaxel in inducing apoptosis in the primary OC cells. Curcumin was not able to affect the carboplatin mediated cell killing. However, a combination of curcumin and paclitaxel was additive and was equally effective as a combination of carboplatin and paclitaxel. A combination of curcumin carboplatin, and paclitaxel was also found to be additive and, in fact, turned out to be the best combination that gave the highest percentage of apoptosis in vitro. CONCLUSION: This study highlights the fact that primary cultures of OC cells can be used to detect response to chemotherapeutic agents and help to individualize the treatment offered to OC patients.