In silico selection of aptamers against SARS-CoV-2.

Aptamers are molecular recognition elements that have been extensively deployed in a wide array of applications ranging from diagnostics to therapeutics. Due to their unique properties as compared to antibodies, aptamers were also largely isolated during the COVID-19 pandemic for multiple purposes. Typically generated by conventional SELEX, the inherent drawbacks of the process including the time-consuming, cumbersome and resource-intensive nature catalysed the move to adopt in silico approaches to isolate aptamers. Impressive performances of these in silico-derived aptamers in their respective assays have been documented thus far, bearing testimony to the huge potential of the in silico approaches, akin to the traditional SELEX in isolating aptamers. In this study, we provide an overview of the in silico selection of aptamers against SARS-CoV-2 by providing insights into the basic steps involved, which comprise the selection of the initial single-stranded nucleic acids, determination of the secondary and tertiary structures and in silico approaches that include both rigid docking and molecular dynamics simulations. The different approaches involving aptamers against SARS-CoV-2 were illuminated and the need to verify these aptamers by experimental validation was also emphasized. Cognizant of the need to continuously improve aptamers, the strategies embraced thus far for post-in silico selection modifications were enumerated. Shedding light on the steps involved in the in silico selection can set the stage for further improvisation to augment the functionalities of the aptamers in the future.

A novel chromone-based as a potential inhibitor of ULK1 that modulates autophagy and induces apoptosis in colon cancer.

Aim: Chromones are promising for anticancer drug development.Methods & results: 12 chromone-based compounds were synthesized and tested against cancer cell lines. Compound 8 showed the highest cytotoxicity (LC50 3.2 µM) against colorectal cancer cells, surpassing 5-fluorouracil (LC50 4.2 µM). It suppressed colony formation, induced cell cycle arrest and triggered apoptotic cell death, confirmed by staining and apoptosis markers. Cell death was accompanied by enhanced reactive oxygen species formation and modulation of the autophagic machinery (autophagy marker light chain 3B (LC3B); adenosine monophosphate-activated protein kinase (AMPK); protein kinase B (PKB); UNC-51-like kinase (ULK)-1; and ULK2). Molecular docking and dynamic simulations revealed that compound 8 directly binds to ULK1.Conclusion: Compound 8 is a promising lead for autophagy-modulating anti-colon cancer drugs.

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Autophagy inhibition suppresses Newcastle disease virus-induced cell death by inhibiting viral replication in human breast cancer cells.

Newcastle disease virus (NDV) is an oncolytic virus which selectively replicates in cancer cells without harming normal cells. Autophagy is a cellular mechanism that breaks down unused cytoplasmic constituents into nutrients. In previous studies, autophagy enhanced NDV-induced oncolysis in lung cancer and glioma cells. However, the effect of autophagy inhibition on NDV-induced oncolysis in breast cancer cells remains unknown. This study aimed to examine the effect of autophagy inhibition on NDV-induced oncolysis in human breast cancer cells, MCF7. To inhibit autophagy, we knocked down the expression of the autophagy protein beclin-1 (BECN1) by short interfering RNA (siRNA). The cells were infected with the recombinant NDV strain AF2240 expressing green fluorescent protein. We found that NDV induced autophagy and knockdown of BECN1 significantly reduced the NDV-induced autophagy in MCF7 cells. Importantly, BECN1 knockdown significantly suppressed cell death by inhibiting viral replication, as observed at 24 h post infection. Overall, our data suggest that autophagy inhibition may not be a suitable strategy to enhance NDV oncolytic efficacy against breast cancer.

Knockdown of the Autophagy Protein Beclin-1 Does Not Affect Innate Cytokine Production in Human Lung Epithelial Cells during Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in young children, globally. Autophagy is a cellular degradation process that mediates cell survival. Studies using mouse models have demonstrated that inhibiting autophagy affects the production of cytokines triggered by RSV. However, the effect of autophagy on RSV-induced cytokine production in human cells remains inadequately studied. Our previous research showed that inhibiting autophagy using pharmacological inhibitors did not affect the innate cytokine production in human lung epithelial cells (BEAS-2B) following RSV infection. In this study, we sought to validate these findings using a more specific approach, employing short-interfering RNA (siRNA) to target the important autophagy protein Beclin-1 (Bec-1). Prior to measuring cytokine production, we confirmed that silencing Bec-1 with siRNA effectively suppressed autophagy without affecting cell viability. Our results revealed that inhibiting autophagy through Bec-1 knockdown did not affect the production of innate cytokines CXCL8 and CCL5 in BEAS-2B cells during RSV infection, consistent with our previous findings using pharmacological inhibitors. Overall, our data suggest that targeting autophagy may not be an effective strategy for alleviating RSV-induced airway inflammation.

Analysis of PPI networks of transcriptomic expression identifies hub genes associated with Newcastle disease virus persistent infection in bladder cancer.

Bladder cancer cells can acquire persistent infection of oncolytic Newcastle disease virus (NDV) but the molecular mechanism(s) remain unelucidated. This poses a major barrier to the effective clinical translation of oncolytic NDV virotherapy of cancers. To improve our understanding of the molecular mechanism(s) associated with the development of NDV persistent infection in bladder cancer, we used mRNA expression profiles of persistently infected bladder cancer cells to construct PPI networks. Based on paths and modules in the PPI network, the bridges were found mainly in the upregulated mRNA-pathways of p53 signalling, ECM-receptor interaction, and TGF-beta signalling and downregulated mRNA-pathways of antigen processing and presentation, protein processing in endoplasmic reticulum, completement and coagulation cascades in persistent TCCSUPPi cells. In persistent EJ28Pi cells, connections were identified mainly through upregulated mRNA-pathways of renal carcinoma, viral carcinogenesis, Ras signalling and cell cycle and the downregulated mRNA-pathways of Wnt signalling, HTLV-I infection and pathways in cancers. These connections were mainly dependent on RPL8-HSPA1A/HSPA4 in TCCSUPPi cells and EP300, PTPN11, RAC1-TP53, SP1, CCND1 and XPO1 in EJ28Pi cells. Oncomine validation showed that the top hub genes identified in the networks that include RPL8, THBS1, F2 from TCCSUPPi and TP53 and RAC1 from EJ28Pi are involved in the development and progression of bladder cancer. Protein-drug interaction networks identified several putative drug targets that could be used to disrupt the linkages between the modules and prevent bladder cancer cells from acquiring NDV persistent infection. This novel PPI network analysis of differentially expressed mRNAs of NDV persistently infected bladder cancer cell lines provide an insight into the molecular mechanisms of NDV persistency of infection in bladder cancers and the future screening of drugs that can be used together with NDV to enhance its oncolytic efficacy.

Discovery of Ruthenium(II) Metallocompound and Olaparib Synergy for Cancer Combination Therapy.

Synergistic drug combinations can extend the use of poly(ADP-ribose) polymerase inhibitors (PARPi) such as Olaparib to BRCA-proficient tumors and overcome acquired or de novo drug resistance. To identify new synergistic combinations for PARPi, we screened a "micro-library" comprising a mix of commercially available drugs and DNA-binding ruthenium(II) polypyridyl complexes (RPCs) for Olaparib synergy in BRCA-proficient triple-negative breast cancer cells. This identified three hits: the natural product Curcumin and two ruthenium(II)-rhenium(I) polypyridyl metallomacrocycles. All combinations identified were effective in BRCA-proficient breast cancer cells, including an Olaparib-resistant cell line, and spheroid models. Mechanistic studies indicated that synergy was achieved via DNA-damage enhancement and resultant apoptosis. Combinations showed low cytotoxicity toward non-malignant breast epithelial cells and low acute and developmental toxicity in zebrafish embryos. This work identifies RPC metallomacrocycles as a novel class of agents for cancer combination therapy and provides a proof of concept for the inclusion of metallocompounds within drug synergy screens.

Discovery of a novel dual functional phenylpyrazole-styryl hybrid that induces apoptotic and autophagic cell death in bladder cancer cells.

Unpleasant side effects and resistance development remained the Achilles heel of chemotherapy. Since low tumor-selectivity and monotonous effect of chemotherapy are closely related to such bottleneck, targeting tumor-selective multi-functional anticancer agents may be an ideal strategy in the search of new safer drugs. Herein, we report the discovery of compound 21, a nitro-substituted 1,5-diphenyl-3-styryl-1H-pyrazole that possesses dual functional characteristics. The 2D- and 3D-culture-based studies revealed that 21 not only could induce ROS-independent apoptotic and EGFR/AKT/mTOR-mediated autophagic cell deaths in EJ28 cells simultaneously but also has the ability in inducing cell death at both proliferating and quiescent zones of EJ28 spheroids. The molecular modelling analysis showed that 21 possesses EGFR targeting capability as it forms stable interactions in the EGFR active site. Together with its good safety profile in the zebrafish-based model, the present study showed that 21 is promising and may lead to the discovery of tumor-selective multi-functional anti-cancer agents.

Synergy of ruthenium metallo-intercalator, [Ru(dppz)2(PIP)]2+, with PARP inhibitor Olaparib in non-small cell lung cancer cells.

Poly(ADP-ribose) polymerase (PARP) are critical DNA repair enzymes that are activated as part of the DNA damage response (DDR). Although inhibitors of PARP (PARPi) have emerged as small molecule drugs and have shown promising therapeutic effects, PARPi used as single agents are clinically limited to patients with mutations in germline breast cancer susceptibility gene (BRCA). Thus, novel PARPi combination strategies may expand their usage and combat drug resistance. In recent years, ruthenium polypyridyl complexes (RPCs) have emerged as promising anti-cancer candidates due to their attractive DNA binding properties and distinct mechanisms of action. Previously, we reported the rational combination of the RPC DNA replication inhibitor [Ru(dppz)2(PIP)]2+ (dppz = dipyrido[3,2-a:2',3'-c]phenazine, PIP = 2-(phenyl)-imidazo[4,5-f][1,10]phenanthroline), "Ru-PIP", with the PARPi Olaparib in breast cancer cells. Here, we expand upon this work and examine the combination of Ru-PIP with Olaparib for synergy in lung cancer cells, including in 3D lung cancer spheroids, to further elucidate mechanisms of synergy and additionally assess toxicity in a zebrafish embryo model. Compared to single agents alone, Ru-PIP and Olaparib synergy was observed in both A549 and H1975 lung cancer cell lines with mild impact on normal lung fibroblast MRC5 cells. Employing the A549 cell line, synergy was confirmed by loss in clonogenic potential and reduced migration properties. Mechanistic studies indicated that synergy is accompanied by increased double-strand break (DSB) DNA damage and reactive oxygen species (ROS) levels which subsequently lead to cell death via apoptosis. Moreover, the identified combination was successfully able to inhibit the growth of A549 lung cancer spheroids and acute zebrafish embryos toxicity studies revealed that this combination showed reduced toxicity compared to single-agent Ru-PIP.

Persistent Newcastle disease virus infection in bladder cancer cells is associated with putative pro-survival and anti-viral transcriptomic changes.

BACKGROUND: Newcastle disease virus (NDV) is an oncolytic virus with excellent selectivity against cancer cells, both in vitro and in vivo. Unfortunately, prolonged in vitro NDV infection results in the development of persistent infection in the cancer cells which are then able to resist NDV-mediated oncolysis. However, the mechanism of persistency of infection remains poorly understood. METHODS: In this study, we established persistently NDV-infected EJ28 bladder cancer cells, designated as EJ28P. Global transcriptomic analysis was subsequently carried out by microarray analysis. Differentially expressed genes (DEGs) between EJ28 and EJ28P cells identified by the edgeR program were further analysed by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) analyses. In addition, the microarray data were validated by RT-qPCR. RESULTS: Persistently NDV-infected EJ28 bladder cancer cells were successfully established and confirmed by flow cytometry. Microarray analysis identified a total of 368 genes as differentially expressed in EJ28P cells when compared to the non-infected EJ28 cells. GSEA revealed that the Wnt/ß-catenin and KRAS signalling pathways were upregulated while the TGF-ß signalling pathway was downregulated. Findings from this study suggest that the upregulation of genes that are associated with cell growth, pro-survival, and anti-apoptosis may explain the survivability of EJ28P cells and the development of persistent infection of NDV. CONCLUSIONS: This study provides insights into the transcriptomic changes that occur and the specific signalling pathways that are potentially involved in the development and maintenance of NDV persistency of infection in bladder cancer cells. These findings warrant further investigation and is crucial towards the development of effective NDV oncolytic therapy against cancer.

Inhibition of Autophagy Does Not Affect Innate Cytokine Production in Human Lung Epithelial Cells During Respiratory Syncytial Virus Infection.

Human respiratory syncytial virus (RSV) is one of the major causes of childhood acute lower respiratory tract infection worldwide. Autophagy is an intracellular pathway involved in nutrient recycling. Recently, autophagy has been reported to play a role in regulating host cytokine response to several viruses, including vesicular stomatitis virus and human immunodeficiency virus. Previous in vivo studies using mouse model has shown that inhibition of autophagy reduces RSV-induced cytokine production. However, the role of autophagy in modulating RSV-induced cytokine response in human cells has not been reported. We investigated the role of autophagy in regulating the production of the cytokines C-X-C motif ligand 8 (CXCL8) and C-C motif ligand 5 (CCL5), in RSV-infected human bronchial epithelium BEAS-2B cells. Fluorescent microscopic analysis showed that RSV infection induced autophagosome formation in BEAS-2B cells. This autophagy inducing ability of RSV was further confirmed by flow cytometry. The effects of pharmacological inhibition of autophagy by SAR405 or chloroquine on cell death and cytokine release were quantified using lactate dehydrogenase assay and enzyme-linked immunosorbent assay (ELISA), respectively. We found that SAR405 or chloroquine did not cause cell death. Importantly, ELISA analysis showed that pharmacological inhibition of autophagy by SAR405 or chloroquine did not affect the productions of both CXCL5 and CXCL8. In contrast to the previous studies using mouse model, our data suggest that pharmacological inhibition of autophagy may not be a suitable strategy in controlling RSV-induced airway inflammation.

Development of a T7 RNA polymerase expressing cell line using lentivirus vectors for the recovery of recombinant Newcastle disease virus.

The development of a T7 RNA polymerase (T7 RNAP) expressing cell line i.e. BSR T7/5 cells marks an improvement of reverse genetics for the recovery of recombinant Newcastle disease virus (rNDV). BSR T7/5 is developed by transient transfection of plasmid encoding T7 RNAP gene for rNDV rescue. However, the gene expression decreases gradually over multiple passages and eventually hinders the rescue of rNDV. To address this issue, lentiviral vector was used to develop T7 RNAP-expressing HEK293-TA (HEK293-TA-Lv-T7) and SW620 (SW620-Lv-T7) cell lines, evidenced by the expression of T7 RNAP after subsequent 20 passages. rNDV was rescued successfully using HEK293-TA-Lv-T7 clones (R1D3, R1D8, R5B9) and SW620-Lv-T7 clones (R1C11, R3C5) by reverse transfection, yielding comparable virus rescue efficiency and virus titres to that of BSR T7/5. This study provides new tools for rNDV rescue and insights into cell line development and virology by reverse genetics.

Synthesis and Optimization of Mesoporous Silica Nanoparticles for Ruthenium Polypyridyl Drug Delivery.

The ruthenium polypyridyl complex [Ru(dppz)2PIP]2+ (dppz: dipyridophenazine, PIP: (2-(phenyl)-imidazo[4,5-f ][1,10]phenanthroline), or Ru-PIP, is a potential anticancer drug that acts by inhibiting DNA replication. Due to the poor dissolution of Ru-PIP in aqueous media, a drug delivery agent would be a useful approach to overcome its limited bioavailability. Mesoporous silica nanoparticles (MSNs) were synthesized via a co-condensation method by using a phenanthrolinium salt with a 16 carbon length chain (Phen-C16) as the template. Optimization of the synthesis conditions by Box-Behnken design (BBD) generated MSNs with high surface area response at 833.9 m2g-1. Ru-PIP was effectively entrapped in MSNs at 18.84%. Drug release profile analysis showed that Ru-PIP is gradually released, with a cumulative release percentage of approximately 50% at 72 h. The release kinetic profile implied that Ru-PIP was released from MSN by diffusion. The in vitro cytotoxicity of Ru-PIP, both free and MSN-encapsulated, was studied in Hela, A549, and T24 cancer cell lines. While treatment of Ru-PIP alone is moderately cytotoxic, encapsulated Ru-PIP exerted significant cytotoxicity upon all the cell lines, with half maximal inhibitory concentration (IC50) values determined by MTT (([3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyltetrazolium bromide]) assay at 48 h exposure substantially decreasing from >30 µM to <10 µM as a result of MSN encapsulation. The mechanistic potential of cytotoxicity on cell cycle distribution showed an increase in G1/S phase populations in all three cell lines. The findings indicate that MSN is an ideal drug delivery agent, as it is able to sustainably release Ru-PIP by diffusion in a prolonged treatment period.

Isolation and Characterization of Novel Phages Targeting Xanthomonas oryzae: Culprit of Bacterial Leaf Blight Disease in Rice.

Background: Bacterial leaf blight (BLB) disease caused 80% of disease incidence in paddy in Kedah and Selangor states of Malaysia. The pathogenic bacterium, Xanthomonas oryzae pv. oryzae (Xoo), is one of the destructive pathogens infecting lowland irrigated and rainfed paddy in Asia's tropical and temperate environments. Bacteriophages (or phages) have been proposed to control the pathogen due to their efficacy and safety aspects. Material and Methods: In this study, a total of 70 Xoo-phages were isolated from termite which living in rice-growing area. Results: 2 lytic phages NΦ-1 and NΦ-3 were selected due to the high titer of the virus. Electron microscopic analysis showed that those phages belonged to the family Podoviridae, order Caudovirales with short noncontracted tails. Moreover, these phages have a narrow host range specifically target Xoo with a higher burst size. Whole-genome sequencing showed that the Xoo-phage NΦ-1 and NΦ-3 consists of a linear double-stranded DNA molecule of length 41,151 and 38,454 bp, respectively. Conclusion: This study successfully characterized two novel Xanthomonas phages and their potential as antimicrobial agents against BLB disease in rice.

Oncolytic effects of the recombinant Newcastle disease virus, rAF-IL12, against colon cancer cells in vitro and in tumor-challenged NCr-Foxn1nu nude mice.

Colon cancer remains one of the main cancers causing death in men and women worldwide as certain colon cancer subtypes are resistant to conventional treatments and the development of new cancer therapies remains elusive. Alternative modalities such as the use of viral-based therapeutic cancer vaccine is still limited, with only the herpes simplex virus (HSV) expressing granulocyte-macrophage colony- stimulating factor (GM-CSF) or talimogene laherparepvec (T-Vec) being approved in the USA and Europe so far. Therefore, it is imperative to continue the search for a new treatment modality. This current study evaluates a combinatorial therapy between the oncolytic Newcastle disease virus (NDV) and interleukin-12 (IL-12) cytokine as a potential therapeutic vaccine to the current anti-cancer drugs. Several in vitro analyses such as MTT assay, Annexin V/FITC flow cytometry, and cell cycle assay were performed to evaluate the cytotoxicity effect of recombinant NDV, rAF-IL12. Meanwhile, serum cytokine, serum biochemical, histopathology of organs and TUNEL assay were carried out to assess the anti-tumoral effects of rAF-IL12 in HT29 tumor-challenged nude mice. The apoptosis mechanism underlying the effect of rAF-IL12 treatment was also investigated using NanoString Gene expression analysis. The recombinant NDV, rAF-IL12 replicated in HT29 colon cancer cells as did its parental virus, AF2240-i. The rAF-IL12 treatment had slightly better cytotoxicity effects towards HT29 cancer cells when compared to the AF2240-i as revealed by the MTT, Annexin V FITC and cell cycle assay. Meanwhile, the 28-day treatment with rAF-IL12 had significantly (p < 0.05) perturbed the growth and progression of HT29 tumor in NCr-Foxn1nu nude mice when compared to the untreated and parental wild-type NDV strain AF2240-i. The rAF-IL12 also modulated the immune system in nude mice by significantly (p < 0.05) increased the level of IL-2, IL-12, and IFN-γ cytokines. Treatment with rAF-IL12 had also significantly (p < 0.05) increased the expression level of apoptosis-related genes such as Fas, caspase-8, BID, BAX, Smad3 and granzyme B in vitro and in vivo. Besides, rAF-IL12 intra-tumoral delivery was considered safe and was not hazardous to the host as evidenced in pathophysiology of the normal tissues and organs of the mice as well as from the serum biochemistry profile of liver and kidney. Therefore, this study proves that rAF-IL12 had better cytotoxicity effects than its parental AF2240-i and could potentially be an ideal treatment for colon cancer in the near future.

In-Vitro and In-Silico Evaluations of Heterocyclic-Containing Diarylpentanoids as Bcl-2 Inhibitors Against LoVo Colorectal Cancer Cells.

In the present study, we investigated the in-vitro anti-cancer potential of six diarylpentanoids against a panel of BRAF- and KRAS-mutated colorectal cancer cell lines including T84, SW620, LoVo, HT29, NCI-H508, RKO, and LS411N cells. Structure-activity relationship study suggested that the insertions of tetrahydro-4H-thiopyran-4-one and brominated phenyl moieties are essential for better cytotoxicity. Among the evaluated analogs, 2e has been identified as the lead compound due to its low IC50 values of approximately 1 µM across all cancer cell lines and high chemotherapeutic index of 7.1. Anti-proliferative studies on LoVo cells showed that 2e could inhibit cell proliferation and colony formations by inducing G2/M cell cycle arrest. Subsequent cell apoptosis assay confirmed that 2e is a Bcl-2 inhibitor that could induce intrinsic cell apoptosis by creating a cellular redox imbalance through its direct inhibition on the Bcl-2 protein. Further molecular docking studies revealed that the bromophenyl moieties of 2e could interact with the Bcl-2 surface pocket through hydrophobic interaction, while the tetrahydro-4H-thiopyran-4-one fragment could form additional Pi-sulfur and Pi-alkyl interactions in the same binding site. In all, the present results suggest that 2e could be a potent lead that deserves further modification and investigation in the development of a new Bcl-2 inhibitor.

Cytotoxicity study of the interleukin-12-expressing recombinant Newcastle disease virus strain, rAF-IL12, towards CT26 colon cancer cells in vitro and in vivo.

BACKGROUND: Oncolytic viruses have emerged as an alternative therapeutic modality for cancer as they can replicate specifically in tumour cells and induce toxic effects leading to apoptosis. Despite the great potentials and promising results shown in multiple studies, it appears that their efficacy is still moderate and deemed as not sufficient in clinical studies. In addressing this issue, genetic/molecular engineering approach has paved its way to improve the therapeutic efficacy as observed in the case of herpes simplex virus (HSV) expressing granulocyte-macrophage colony-stimulating factor (GM-CSF). This study aimed to explore the cytotoxicity effects of recombinant NDV strain AF2240-i expressing interleukin-12 (rAF-IL12) against CT26 colon cancer cells. METHODS: The cytotoxicity effect of rAF-IL12 against CT26 colon cancer cell line was determined by MTT assay. Based on the IC50 value from the anti-proliferative assay, further downward assays such as Annexin V FITC and cell cycle progression were carried out and measured by flow cytometry. Then, the in vivo study was conducted where the rAF-IL12 viral injections were given at the intra-tumoral site of the CT26 tumour-burden mice. At the end of the experiment, serum biochemical, T cell immunophenotyping, serum cytokine, histopathology of tumour and organ section, TUNEL assay, and Nanostring gene expression analysis were performed. RESULTS: The rAF-IL12 induced apoptosis of CT26 colon cancer cells in vitro as revealed in the Annexin V FITC analysis and also arrested the cancer cells progression at G1 phase of the cell cycle analysis. On the other hand, the rAF-IL12 significantly (p < 0.05) inhibited the growth of CT26 tumour in Balb/c mice and had regulated the immune system by increasing the level of CD4 + , CD8 + , IL-2, IL-12, and IFN-γ. Furthermore, the expression level of apoptosis-related genes (bax and p53) was up-regulated as a result of the rAF-IL12 treatment. Additionally, the rAF-IL12 had also down-regulated the expression level of KRAS, BRAF, MAPK1, Notch1, CCL2, and VEGF oncogenes. Besides, rAF-IL12 intra-tumoral delivery was considered safe and not hazardous to the host as evidenced in pathophysiology of the normal tissues and organs of the mice as well as from the serum biochemistry profile of liver and kidney. CONCLUSIONS: These results indicated that rAF-IL12 had better anti-tumoral and cytotoxicity effects compared to its parental wild-type, AF2240-i in combatting the CT26 colon cancer model.

Synthesis and in-vitro anti-cancer evaluations of multi-methoxylated asymmetrical diarylpentanoids as intrinsic apoptosis inducer against colorectal cancer.

In the present study, a series of nine stable 3,4,5-methoxylphenyl-containing asymmetrical diarylpentanoids, derivatives of curcuminoids, have been synthesized, characterized and evaluated for their in-vitro anti-cancer potential against a panel of BRAF- and KRAS-mutated colorectal cancer cell lines including T84, LoVo and SW620, HT29, RKO and NCI-H508, respectively. Structure-activity relationship study on cytotoxicity of tested compounds suggested that the presence of meta-hydroxyl and adjacent dimethoxyl groups are crucial for enhanced cytotoxicity of diarylpentanoids. Among the evaluated analogs, 8 has been identified as the lead compound due to its highest chemotherapeutic index of 9.9 and nano molar scale cytotoxicity against SW620 and RKO. Colonies formation and cell cycle analyses on 8-treated RKO cells showed that 8 exhibits strong anti-proliferative activity by inducing G2/M-phase cell arrest. Subsequent flow cytometry based annexin-V and DCFHDA studies suggested that 8 could induce apoptosis through intracellular ROS-dependent pathway. Further Western blot studies confirmed that 8 has induced intrinsic apoptosis in RKO cells through the up-regulations of Bad and Bax pro-apoptotic proteins and down-regulations of Bcl-2 and Bcl-xL pro-survival proteins. In all, the present results suggest that 8 could be a potent lead which deserves further modification and investigation in the development of small molecule-based anti-colorectal cancer agents.

An improved method for the rescue of recombinant Newcastle disease virus.

Reverse genetics has been used to generate recombinant Newcastle disease virus with enhanced immunogenic properties for vaccine development. The system, which involves co-transfecting the viral antigenomic plasmid with three helper plasmids into a T7 RNA polymerase-expressing cell to produce viral progenies, poses a great challenge. We have modified the standard transfection method to improve the transfection efficiency of the plasmids, resulting in a higher titer of virus progeny production. Two transfection reagents (i.e., lipofectamine and polyethylenimine) were used to compare the transfection efficiency of the four plasmids. The virus progenies produced were quantitated with flow cytometry analysis of the infectious virus unit. The modified transfection method increased the titer of virus progenies compared with that of the standard transfection method.

Metallointercalator [Ru(dppz)2(PIP)]2+ Renders BRCA Wild-Type Triple-Negative Breast Cancer Cells Hypersensitive to PARP Inhibition.

There is a need to improve and extend the use of clinically approved poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi), including for BRCA wild-type triple-negative breast cancer (TNBC). The demonstration that ruthenium(II) polypyridyl complex (RPC) metallointercalators can rapidly stall DNA replication fork progression provides the rationale for their combination alongside DNA damage response (DDR) inhibitors to achieve synergism in cancer cells. The aim of the present study was to evaluate use of the multi-intercalator [Ru(dppz)2(PIP)]2+ (dppz = dipyrido[3,2-a:2',3'-c]phenazine, PIP = (2-(phenyl)imidazo[4,5-f][1,10]phenanthroline, Ru-PIP) alongside the PARPi olaparib and NU1025. Cell proliferation and clonogenic survival assays indicated a synergistic relationship between Ru-PIP and olaparib in MDA-MB-231 TNBC and MCF7 human breast cancer cells. Strikingly, low dose Ru-PIP renders both cell lines hypersensitive to olaparib, with a >300-fold increase in olaparib potency in TNBC, the largest nongenetic PARPi enhancement effect described to date. A negligible impact on the viability of normal human fibroblasts was observed for any combination tested. Increased levels of DNA double-strand break (DSB) damage and olaparib abrogation of Ru-PIP-activated pChk1 signaling are consistent with PARPi-facilitated collapse of Ru-PIP-associated stalled replication forks. This results in enhanced G2/M cell-cycle arrest, apoptosis, and decreased cell motility for the combination treatment compared to single-agent conditions. This work establishes that an RPC metallointercalator can be combined with PARPi for potent synergy in BRCA-proficient breast cancer cells, including TNBC.

Evaluation of a Recombinant Newcastle Disease Virus Expressing Human IL12 against Human Breast Cancer.

The Newcastle disease virus (NDV) strain AF2240 is an avian avulavirus that has been demonstrated to possess oncolytic activity against cancer cells. However, to illicit a greater anti-cancer immune response, it is believed that the incorporation of immunostimulatory genes such as IL12 into a recombinant NDV backbone will enhance its oncolytic effect. In this study, a newly developed recombinant NDV that expresses IL12 (rAF-IL12) was tested for its safety, stability and cytotoxicity. The stability of rAF-IL12 was maintained when passaged in specific pathogen free (SPF) chicken eggs from passage 1 to passage 10; with an HA titer of 29. Based on the results obtained from the MTT cytotoxic assay, rAF-IL12 was determined to be safe as it only induced cytotoxic effects against normal chicken cell lines and human breast cancer cells while sparing normal cells. Significant tumor growth inhibition (52%) was observed in the rAF-IL12-treated mice. The in vivo safety profile of rAF-IL12 was confirmed through histological observation and viral load titer assay. The concentration and presence of the expressed IL12 was quantified and verified via ELISA assay. In summary, rAF-IL12 was proven to be safe, selectively replicating in chicken and cancer cells and was able to maintain its stability throughout several passages; thus enhancing its potential as an anti-breast cancer vaccine.