Scaffold overlay of flavonoid-inspired molecules: Discovery of 2,3-diaryl-pyridopyrimidin-4-imine/ones as dual hTopo-II and tubulin targeting anticancer agents.

Almost half of all medicines approved by the U.S. Food and Drug Administration have been found to be developed based on inspiration from natural products (NPs). Here, we report a novel strategy of scaffold overlaying of scaffold-hopped analogs of bioactive flavones and isoflavones and installation of drug-privileged motifs, which has led to discovery of anticancer agents that surpass the functional efficiency of the original NPs. The analogs, 2,3-diaryl-pyridopyrimidin-4-imine/ones were efficiently synthesized by an approach of a nitrile-stabilized quaternary ammonium ylide as masked synthon and Pd-catalyzed activation-arylation methods. Compared to the NPs, these NP-analogs exhibited differentiated functions; dual inhibition of human topoisomerase-II (hTopo-II) enzyme and tubulin polymerization, and pronounced antiproliferative effect against various cancer cell lines, including numerous drug-resistant cancer cells. The most active compound 5l displayed significant inhibition of migration ability of cancer cells and blocked G1/S phase transition in cell cycle. Compound 5l caused pronounced effect in expression patterns of various key cell cycle regulatory proteins; up-regulation of apoptotic proteins, Bax, Caspase 3 and p53, and down-regulation of apoptosis-inhibiting proteins, BcL-xL, Cyclin D1, Cyclin E1 and NF-κB, which indicates high efficiency of the molecule 5l in apoptosis-signal axis interfering potential. Cheminformatics analysis revealed that 2,3-diaryl-pyridopyrimidin-4-imine/ones occupy a distinctive drug-relevant chemical space that is seldom represented by natural products and good physicochemical, ADMET and pharmacokinetic-relevant profile. Together, the anticancer potential of the investigated analogs was found to be much more efficient compared to the original natural products and two anticancer drugs, Etoposide (hTopo-II inhibitor) and 5-Flurouracile (5-FU).

Design, synthesis, and anticancer activity of some novel 1H-benzo[d]imidazole-5-carboxamide derivatives as fatty acid synthase inhibitors.

Multiple malignancies exhibit aberrant FASN expression, associated with enhanced de novo lipogenesis to meet the metabolic demands of rapidly proliferating tumour cells. Furthermore, elevated FASN expression has been linked to tumour aggressiveness and poor prognosis in a variety of malignant tumours, making FASN is an attractive target for anticancer drug discovery. Herein, we report the de novo design and synthesis of (2-(2-hydroxyphenyl)-1H-benzo[d]imidazol-5-yl)(piperazin-1-yl)methanone derivatives as novel FASN inhibitors with potential therapeutic applications in breast and colorectal cancers. Twelve (2-(2-hydroxyphenyl)-1H-benzo[d]imidazol-5-yl)(piperazin-1-yl)methanone derivatives (CTL) were synthesized and evaluated for FASN inhibition and cytotoxicity against colon cancer (HCT-116, Caco-2 cell lines), breast cancer (MCF-7 cell line) and normal cell line (HEK-293). Compounds CTL-06 and CTL-12 were chosen as the most promising lead molecules based on FASN inhibition and selective cytotoxicity profiles against colon and breast cancer cell lines. Compounds CTL-06 and CTL-12 demonstrate promising FASN inhibitory activity at IC50 of 3 ± 0.25 µM and 2.5 ± 0.25 µM when compared to the FASN inhibitor orlistat, which has an IC50 of 13.5 ± 1.0 µM. Mechanistic investigations on HCT-116 revealed that CTL-06 and CTL-12 treatment led to cell cycle arrest in Sub-G1/S phase along with apoptosis induction. Western blot studies indicated that CTL-06 and CTL-12 inhibited FASN expression in a dose-dependent manner. CTL-06 and CTL-12 treatment of HCT-116 cells enhanced caspase-9 expression in a dose-dependent manner, while upregulating proapoptotic marker Bax and downregulating antiapoptotic Bcl-xL. Molecular docking experiments of CTL-06 and CTL-12 with FASN enzyme revealed the mode of binding of these analogues in the KR domain of the enzyme.

Olaparib enhances the Resveratrol-mediated apoptosis in breast cancer cells by inhibiting the homologous recombination repair pathway.

Although sensitization of BRCA-mutated, homologous recombination (HR)-deficient breast cancer cells through PARP inhibitor is widely studied, not much is known about the treatment of BRCA-wild-type, HR-proficient breast cancer. Here, we aim to investigate whether a bioactive compound, Resveratrol (RES), can induce DNA double-strand breaks in HR-proficient breast cancer cells and Olaparib (OLA), a PARP inhibitor, can enhance the RES-mediated apoptosis by deregulating the HR repair pathway. The detailed mechanism of anti-cancer action of RES + OLA combination in breast cancer has been evaluated using in vitro, ex vivo, and in vivo preclinical model systems. OLA increased RES-mediated DNA damage, downregulated the HR pathway proteins, caused a late S/G2 cell cycle arrest, enhanced apoptosis and cell death in RES pre-treated breast cancer cells at much lower concentrations than their individual treatments. Direct measurement of HR pathway activity using a GFP plasmid-based assay demonstrated reduced HR efficiency in I-SceI endonuclease-transfected cells treated with OLA. Moreover, RES + OLA treatment also caused significant reduction in PARP1-mediated PARylation and efficiently trapped PARP1 at the DNA damage site. Upon RES treatment, PARylated PARP1 was found to interact with BRCA1, which then activated other HR pathway proteins. But after addition of OLA in RES pre-treated cells, PARP1 could not interact with BRCA1 due to inhibition of PARylation. This resulted in deregulation of HR pathway. To further confirm the role of BRCA1 in PARP1-mediated HR pathway activation, BRCA1 was knocked down that caused complete inhibition of HR pathway activity, and further enhanced apoptosis after RES + OLA treatment in BRCA1-silenced cells. In agreement with in vitro data, similar experimental results were obtained in ex vivo patient-derived breast cancer cells and in vivo xenograft mice. Thus, RES + OLA combination treatment enhanced breast cancer cell death by causing excessive DNA damage and also simultaneously inhibiting the HR pathway.

TGF-ß1 induced autophagy in cancer associated fibroblasts during hypoxia contributes EMT and glycolysis via MCT4 upregulation.

The Transforming growth factor-ß1 (TGF- ß1) in the tumor microenvironment (TME) is the major cytokine that acts as a mediator of tumor-stroma crosstalk, which in fact has a dual role in either promoting or suppressing tumor development. The cancer-associated fibroblasts (CAFs) are the major cell types in the TME, and the interaction with most of the epithelial cancers is the prime reason for cancer survival. However, the molecular mechanisms, associated with the TGF- ß1 induced tumor promotion through tumor-CAF crosstalk are not well understood. In the Reverse Warburg effect, CAFs feed the adjacent cancer cells by lactate produced during the aerobic glycolysis. We hypothesized that the monocarboxylate transporter, MCT4 which is implicated in lactate efflux from the CAFs, must be overexpressed in the CAFs. Contextually, to explore the role of TGF- ß1 in the hypoxia-induced autophagy in CAFs, we treated CoCl2 and external TGF- ß1 to the human dermal fibroblasts and L929 murine fibroblasts. We demonstrated that hypoxia accelerated the TGF- ß1 signaling and subsequent transformation of normal fibroblasts to CAFs. Moreover, we elucidated that synergistic induction of autophagy by hypoxia and TGF- ß1 upregulate the aerobic glycolysis and MCT4 expression in CAFs. Furthermore, we showed a positive correlation between glucose consumption and MCT4 expression in the CAFs. Autophagy was also found to be involved in the EMT in hypoxic CAFs. Collectively, these findings reveal the unappreciated role of autophagy in TME, which enhances the CAF transformation and that promotes tumor migration and metastasis via the reverse Warburg effect.

PARP inhibitor BMN-673 induced apoptosis by trapping PARP-1 and inhibiting base excision repair via modulation of pol-ß in chromatin of breast cancer cells.

PARP inhibitors emerged as clinically effective anti-tumor agents in combination with DNA damaging agents but the toxicity of DNA damaging agents and their off-target effects caused serious problems in cancer therapy. They confer cytotoxicity in cancer cells both by catalytic inhibition and trapping of PARP-1 at the DNA damage site. There is a lack of direct evidence to quantitatively determine the trapped PARP-1 in cellular DNA. Here, we have precisely evaluated the mechanism of PARP trapping mediated anti-cancer action of Quinacrine (QC), BMN-673, and their combination (QC + BMN-673) in breast cancer cells. We introduced a strategy to measure the cellular PARP trapping potentiality of BMN-673 in QC pretreated cells using a fluorescence-based assay system. It was found that QC+ BMN-673 induced apoptosis by triggering DNA damage in breast cancer cells. Treatment with QC + BMN-673 stimulated the expression of PARP-1 in the chromatin compared to that of PARP-2 and PARP-3. QC + BMN-673 treatment also caused a dose-dependent and time-dependent accumulation of PARP-1 and inhibition of PARylation in the chromatin. Upregulation of BER components (pol-ß and FEN-1), an unchanged HR and NHEJ pathway proteins, and reduction of luciferase activity of the cells transfected with R-p21-P (LP-BER) were noted in combined drug-treated cells. Interestingly, silencing of pol-ß resulted in unchanged PARP-1 trapping and PAR activity in the chromatin with increasing time after QC + BMN-673 treatment without altering APC and FEN-1 expression. Thus, our data suggested that the QC + BMN-673 augmented breast cancer cell death by pol-ß mediated repair inhibition primarily through trapping of PARP-1 besides PARP-1 catalytic inhibition.

Targeting cancer stem cells in the tumor microenvironment: An emerging role of PARP inhibitors.

Cancer stem cells (CSCs) constitute a small population of cancer cells in the tumor microenvironment (TME), which are responsible for metastasis, angiogenesis, drug resistance, and cancer relapse. Understanding the key signatures and resistance mechanisms of CSCs may help in the development of novel chemotherapeutic strategies to specifically target CSCs in the TME. PARP inhibitors (PARPi) are known to enhance the chemosensitivity of cancer cells to other chemotherapeutic agents by inhibiting the DNA repair pathways and chromatin modulation. But their effects on CSCs are still unknown. Few studies have reported that PARPi can stall replication fork progression in CSCs. PARPi also have the potential to overcome chemoresistance in CSCs and anti-angiogenic potentiality as well. Previous reports have suggested that epigenetic drugs can synergistically ameliorate the anti-cancer activities of PARPi through epigenetic modulations. In this review, we have systematically discussed the effects of PARPi on different DNA repair pathways with respect to CSCs and also how CSCs can be targeted either as monotherapy or as a part of combination therapy. We have also talked about how PARPi can help in reversal of chemoresistance of CSCs and the role of PARPi in epigenetic modifications to hinder cancer progression. We have also elaborated on the aspects of research that need to be investigated for development of successful therapeutic interventions using PARPi to specifically target CSCs in the TME.

Organocatalyzed umpolung addition for synthesis of heterocyclic-fused arylidene-imidazolones as anticancer agents.

A strategy of "Nature-to-new" with iterative scaffold-hopping was considered for investigation of privileged ring/functional motif-elaborated analogs of natural aurones. An organocatalyzed umpolung chemistry based method was established for molecular-diversity feasible synthesis of title class of chemotypes i.e. (Z)-2-Arylideneimidazo[1,2-a]pyridinones and (Z)-2-Arylidenebenzo[d]imidazo[2,1-b]thiazol-3-ones. Various biophysical experiments indicated their important biological properties. The analogs showed characteristic anticancer activities with efficiency more than an anticancer drug. The compounds induced apoptosis with arrest in the S phase of the cell cycle regulation. The compounds' significant effect in up/down-regulation of various apoptotic proteins, an apoptosis cascade, and the inhibition of topoisomerases-mediated DNA relaxation process was identified. The analysis of the structure-activity relationship, interference with biological events and the drug-likeness physicochemical properties of the compounds in the acceptable window indicated distinctive medicinal molecule-to-properties of the investigated chemotypes.

NIR irradiation enhances the apoptotic potentiality of quinacrine-gold hybrid nanoparticles by modulation of HSP-70 in oral cancer stem cells.

Cancer stem cells (CSCs) are the tumor cell subpopulations that can self-renew, differentiate, initiate and maintain tumor growth. CSCs are frequently drug-resistant, resulting in tumor recurrence, metastasis, and angiogenesis. Herein, using in vitro oral squamous cell carcinoma (OSCC) CSCs and in vivo xenograft mice model, we have systematically studied the apoptotic potentiality of quinacrine-gold hybrid nanoparticle (QAuNP) and its underlying mechanism after NIR irradiation. QAuNP + NIR caused DNA damage and induced apoptosis in SCC-9-CSCs by deregulating mitochondrial membrane potential (ΔΨm) and activation of ROS. Upregulation of CASPASE-3 and DR-5/DR-4 and reduction of heat shock protein (HSP-70) up to 5-fold were also noticed upon the treatment. The increased expression of DR-5 and CASPASE-3 and decreased expression of HSP-70, CD-44 and Ki-67 were also noted in the xenograft mice treated with QAuNP + NIR + TRAIL. Thus, data suggest that the combined treatment enhances apoptosis in OSCC-CSCs by modulating HSP-70 in the DISC.

Exploration of Benzo[b]carbazole-6,11-diones as anticancer agents: Synthesis and studies of hTopoIIα inhibition and apoptotic effects.

Two series of (hetero)arylamino-naphthoquinones and benzo-fused carbazolequinones were considered for study with the rationale that related structural motifs are present in numerous drugs, clinical trial agents, natural products and hTopoIIα inhibitors. Total 42 compounds were synthesized by reactions including dehydrogenative CN and Pd-catalyzed CC bond forming transformations. These compounds were screened against numerous cancer cells including highly metastatic one (MCF-7, MDA-MB-231, H-357 and HEK293T), and normal cells (MCF 10A). Some of the active compounds were evaluated for clonogenic cell survival and apoptotic effects in cancer cells (DAPI nuclear staining, Comet assay, Annexin-V-FITC/PI dual staining, flow cytometry, and western blot analysis with relevant proteins). All compounds were tested for hTopoIIα inhibitory activity. The investigated series compounds showed important properties like significant apoptotic antiproliferation in cancer cells with cell cycle arrest at S-phase and downregulation of NF- κß signaling cascade, relatively less cytotoxicity to normal cells, and hTopoIIα inhibition with more efficiency compared to an anticancer drug etoposide.

Novel, selective acrylamide linked quinazolines for the treatment of double mutant EGFR-L858R/T790M Non-Small-Cell lung cancer (NSCLC).

T790M mutation is the most common mechanism of acquired resistance to first-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). To overcome this resistance, 4-anilinoquinazoline-based irreversible inhibitors afatinib, dacomitinib has been developed. However, the clinical application of these irreversible inhibitors is limited due to its narrow selectivity against L858R/T790M mutant EGFR. In an attempt to develop potent and selective EGFR T790M inhibitors, we have designed and synthesized two series of novel acrylamide linked quinazolines. Among them, compounds 2i (IC50 0.171 µM) and 11h (IC50 0.159 µM) were identified as potent compounds, which displayed selective and potent anti-proliferative activity on gefitinib-resistant cell line NCI-H1975 as compared to the gefitinib and WZ4002 in cellular assay. Furthermore, a molecular dynamic simulation of 11h was carried out to assess the stability to form a complex with the L858R/T790M EGFR Kinase domain, which demonstrated that complex was stable for the 100 ns and form strong crucial covalent binding contacts with the thiol group of Cys797 residue. Finally, satisfactory in silico pharmacokinetics properties of 2i, 11h and 11i compounds were predicted. The synthesized compounds were also evaluated for in vitro cytotoxic activity/hepatotoxicity against HepG2 cell line through MTT assay. The results revealed that compounds exhibited lower cytotoxicity to HepG2 cells.

Quinacrine Based Gold Hybrid Nanoparticles Caused Apoptosis through Modulating Replication Fork in Oral Cancer Stem Cells.

The presence of cancer stem cells (CSCs) in the tumor microenvironment is responsible for the development of chemoresistance and recurrence of cancer. Our previous investigation revealed the anticancer mechanism of quinacrine-based silver and gold hybrid nanoparticles (QAgNP and QAuNP) in oral cancer cells, but to avoid cancer recurrence, it is important to study the effect of these nanoparticles (NPs) on CSCs. Here, we developed an in vitro CSCs model using SCC-9 oral cancer cells and validated via FACS analysis. Then, 40-60% of cells were found to be CD44+/CD133+ and CD24-. QAuNP showed excellent anti-CSC growth potential against SCC-9-cancer stem like cells (IC50 = 0.4 µg/mL) with the down-regulation of representative CSC markers. Prolonged exposure of QAuNP induced the S-phase arrest and caused re-replication shown by the extended G2/M population and apoptosis to SCC-9-CSC like cells. Up-regulation of BAX, PARP cleavage, and simultaneous down-regulation of Bcl-xL in prolonged treatment to CSCs suggested that the majority of the cells have undergone apoptosis. QAuNP treatment also caused a loss in DNA repair in CSCs. Mostly, the base excision repair (BER) components (Fen-1, DNA ligase-1, Pol-ß, RPA, etc.) were significantly down-regulated after QAuNP treatment, which suggested its action against DNA repair machinery. The replication fork maintenance-related proteins, RAD 51 and BRCA-2, were also deregulated. Very surprisingly, depletion of WRN (an interacting partner for Pre-RC and Fen-1) and a significant increase in expression of fork-degrading nuclease MRE-11 in 96 h treated NPs were observed. Results suggest QAuNP treatment caused excessive DNA damage and re-replication mediated replication stress (RS) and stalling of the replication fork. Inhibition of BER components hinders the flap clearance activity of Fen-1, and it further caused RS and stopped DNA synthesis. Overall, QAuNP treatment led to irreparable replication fork movement, and the stalled replication fork might have degraded by MRE-11, which ultimately results in apoptosis and the death of the CSCs.

Comparative and Mechanistic Study on the Anticancer Activity of Quinacrine-Based Silver and Gold Hybrid Nanoparticles in Head and Neck Cancer.

Using oral cancer cells ( in vitro) and in vivo xenograft mice model, we have systematically studied the detailed mechanism of anticancer activity of quinacrine-based hybrid silver (QAgNP) and gold (QAuNP) nanoparticles (NPs) and compared their efficacies. Both the NPs showed characteristic anti-cell proliferation profile in various cancer cells with minimally affecting the normal nontransformed breast epithelial MCF-10A cells. The IC50 values of QAuNP in various cancer cells were less compared to QAgNP and also found to be the lowest (0.5 µg/mL) in SCC-9 oral cancer cells. Although both NPs caused apoptosis by increased DNA damage, arresting at S phase and simultaneously inhibiting the DNA repair activity in cells, efficacy of QAuNP was better than that of QAgNP. NPs intercalated with DNA and inhibited the topoisomerase activity in cells. Alteration in expression of cell cycle regulatory (cyclins B1, E1, A2, etc.) and replication-related (MRE11, RPA, RFC, etc.) proteins were also observed after NP exposure to the cells. Accumulation of cells resulted in extended G/M phase after prolonged exposure of QAuNP in SCC-9 cells. Interestingly, depletion of geminin and increase of Cdt-1 along with CDC-6 suggest the formation of re-replication. Recovery of body weight and reduction in tumor volume were found in NP-treated xenograft mice. Induction of Bax/Bcl-xL, PARP-1 cleavage, p53, and p21 were noted in NP-treated xenograft mice tissue samples. Thus, data suggest that NP inhibits topoisomerase activity, thereby inhibiting DNA replication and inducing re-replication, which causes S-phase arrest, DNA damage, and finally apoptosis of the oral cancer cells. Also, it was found that anticancer activity of QAuNP is better than that of QAgNP.

Metallic gold and bioactive quinacrine hybrid nanoparticles inhibit oral cancer stem cell and angiogenesis by deregulating inflammatory cytokines in p53 dependent manner.

Complete eradication of aggressive oral cancer remains a challenge due to the presence of CSCs. They resist conventional chemotherapeutic agents due to their self-renewal, drug efflux, and efficient DNA repair capacity. Here, we formulated a hybrid-nanoparticle (QAuNP) using quinacrine and gold and characterized/investigated its anti-angiogenic and anti-metastatic effect on OSCC-CSCs. QAuNP significantly inhibited cellular proliferation, caused apoptosis in vitro, and disrupted angiogenesis in vivo and tumor regression in xenograft mice model. It not only inhibited crucial angiogenic markers Ang-1, Ang-2 and VEGF but also depleted MMP-2 in H-357-PEMT cells in a p53 and p21-dependent manner. QAuNP also increased the ROS and NO generation in OSCC-CSCs and reduced the mitochondrial membrane potential. It altered the level of inflammatory cytokines IL-6, IL-1ß, TNF-α and metastasis-associated markers (CD-44, CD-133) in H-357-PEMT and CM-treated endothelial cells (HUVEC) in p53/p21-dependent manner. Therefore, QAuNP will be a useful therapeutic agent against metastatic OSCC.

Etoposide and doxorubicin enhance the sensitivity of triple negative breast cancers through modulation of TRAIL-DR5 axis.

Death receptor 5 (DR5) is an important target for development of anticancer agents against triple-negative breast cancer (TNBC). Recently, we reported the molecular level details for the modulation of TRAIL-DR5 axis by quinacrine (QC) in breast cancer cells. In this work, the DR5 mediated anticancer potential of topoisomerase inhibitor etoposide (ET) and doxorubicin (DOX) against TNBC has been evaluated. ET and DOX enhanced the DR5 expression in TNBC cells, whereas non-topoisomerase inhibitors pifithrin-α (PIF) and dexamethasone (DEX) failed to do so. In the TRAIL pre-treated cells, ET and DOX induced higher apoptosis, indicating their synergistic effect with TRAIL. The molecular docking and molecular dynamics studies showed their ability to stabilize the TRAIL-DR5 complex, whereas PIF and DEX failed to do so. The binding energy for TRAIL-DR5 complexation in the ternary complexes containing ET (-111.08 kcal/mol) and DOX (-76.35 kcal/mol) were higher than reported binding energy of binary complex (-53.70 kcal/mol). The in silico and in vitro mutational studies highlighted the importance of DR5 residue SerB68 in mediating the receptor-drug interaction. ET and DOX failed to enhance apoptosis in DR5 knockdown (DR5-KD) cells. On the other hand, TRAIL+ET exhibited induction of DR5 and subsequent apoptosis in WT-DR5 overexpressed DR5-KD cells, by modulating the mitochondrial intrinsic apoptosis cascade. An induction of apoptosis and DR5 expression was noticed in xenograft mice and in TNBC patient-derived metastatic cells after TRAIL+ET treatment. Thus, data suggests ET and DOX act as DR5 agonistic ligands and enhance the cellular apoptosis in TNBC.

Nanoquinacrine caused apoptosis in oral cancer stem cells by disrupting the interaction between GLI1 and ß catenin through activation of GSK3ß.

Presences of cancer stem cells (CSCs) in a bulk of cancer cells are responsible for tumor relapse, metastasis and drug resistance in oral cancer. Due to high drug efflux, DNA repair and self-renewable capacity of CSCs, the conventional chemotherapeutic agents are unable to kill the CSCs. CSCs utilizes Hedgehog (HH-GLI), WNT-ß catenin signalling for its growth and development. GSK3ß negatively regulates both the pathways in CSCs. Here, we have shown that a nano-formulated bioactive small molecule inhibitor Quinacrine (NQC) caused apoptosis in oral cancer stem cells (OCSCs; isolated from different oral cancer cells and oral cancer patient derived primary cells) by down regulating WNT-ß catenin and HH-GLI components through activation of GSK3ß. NQC activates GSK3ß in transcriptional and translational level and reduces ß catenin and GLI1 as well as downstream target gene of both the pathways Cyclin D1, C-Myc. The transcription factor activity of both the pathways was also reduced by NQC treatment. GSK3ß, ß catenin and GLI1 interacts with each other and NQC disrupts the co-localization and interaction between ß catenin and GLI1 in OCSCs in a dose dependent manner through activation of GSK3ß. Thus, data suggest NQC caused OCSCs death by disrupting the crosstalk between ß catenin and GLI1 by activation of GSK3ß.

Enhancement of Cytotoxicity and Inhibition of Angiogenesis in Oral Cancer Stem Cells by a Hybrid Nanoparticle of Bioactive Quinacrine and Silver: Implication of Base Excision Repair Cascade.

A poly(lactic-co-glycolic acid) (PLGA)-based uniform (50-100 nm) hybrid nanoparticle (QAgNP) with positive zeta potential (0.52 ± 0.09 mV) was prepared by single emulsion solvent evaporation method with bioactive small molecule quinacrine (QC) in organic phase and silver (Ag) in aqueous phase. Physiochemical properties established it as a true hybrid nanoparticle and not a mixture of QC and Ag. Antitumor activity of QAgNP was evaluated by using various cancer cell lines including H-357 oral cancer cells and OSCC-cancer stem cell in an in vitro model system. QAgNP caused more cytotoxicity in cancer cells than normal epithelial cells by increasing BAX/BCL-XL, cleaved product PARP-1, and arresting the cells at S phase along with DNA damage. In addition, QAgNPs offered greater ability to kill the OSCC-CSCs compared to NQC and AgNPs. QAgNP offered anticancer action in OSCC-CSCs by inhibiting the base excision repair (BER) within the cells. Interestingly, alteration of BER components (Fen-1 and DNA polymerases (ß, δ, and ε) and unalteration of NHEJ (DNA-PKC) or HR (Rad-51) components was noted in QAgNP treated OSCC-CSC cells. Furthermore, QAgNP significantly reduced angiogenesis in comparison to physical mixture of NQC and AgNP in fertilized eggs. Thus, these hybrid nanoparticles caused apoptosis in OSCC-CSCs by inhibiting the angiogenesis and BER in cells.

Resveratrol mediated cell death in cigarette smoke transformed breast epithelial cells is through induction of p21Waf1/Cip1 and inhibition of long patch base excision repair pathway.

Cigarette smoking is a key factor for the development and progression of different cancers including mammary tumor in women. Resveratrol (Res) is a promising natural chemotherapeutic agent that regulates many cellular targets including p21, a cip/kip family of cyclin kinase inhibitors involved in DNA damage-induced cell cycle arrest and blocking of DNA replication and repair. We have recently shown that cigarette smoke condensate (CSC) prepared from commercially available Indian cigarette can cause neoplastic transformation of normal breast epithelial MCF-10A cell. Here we studied the mechanism of Res mediated apoptosis in CSC transformed (MCF-10A-Tr) cells in vitro and in vivo. Res mediated apoptosis in MCF-10A-Tr cells was a p21 dependent event. It increased the p21 protein expression in MCF-10A-Tr cells and MCF-10A-Tr cells-mediated tumors in xenograft mice. Res treatment reduced the tumor size(s) and expression of anti-apoptotic proteins (e.g. PI3K, AKT, NFκB) in solid tumor. The expressions of cell cycle regulatory (Cyclins, CDC-2, CDC-6, etc.), BER associated (Pol-ß, Pol-δ, Pol-ε, Pol-η, RPA, Fen-1, DNA-Ligase-I, etc.) proteins and LP-BER activity decreased in MCF-10A-Tr cells but remain significantly unaltered in isogenic p21 null MCF-10A-Tr cells after Res treatment. Interestingly, no significant changes were noted in SP-BER activity in both the cell lines after Res exposure. Finally, it was observed that increased p21 blocks the LP-BER in MCF-10A-Tr cells by increasing its interaction with PCNA via competing with Fen-1 after Res treatment. Thus, Res caused apoptosis in CSC-induced cancer cells by reduction of LP-BER activity and this phenomenon largely depends on p21.

The contribution of heavy metals in cigarette smoke condensate to malignant transformation of breast epithelial cells and in vivo initiation of neoplasia through induction of a PI3K-AKT-NFκB cascade.

Cigarette smoking is a crucial factor in the development and progression of multiple cancers including breast. Here, we report that repeated exposure to a fixed, low dose of cigarette smoke condensate (CSC) prepared from Indian cigarettes is capable of transforming normal breast epithelial cells, MCF-10A, and delineate the biochemical basis for cellular transformation. CSC transformed cells (MCF-10A-Tr) were capable of anchorage-independent growth, and their anchorage dependent growth and colony forming ability were higher compared to the non-transformed MCF-10A cells. Increased expression of biomarkers representative of oncogenic transformation (NRP-1, Nectin-4), and anti-apoptotic markers (PI3K, AKT, NFκB) were also noted in the MCF-10A-Tr cells. Short tandem repeat (STR) profiling of MCF-10A and MCF-10A-Tr cells revealed that transformed cells acquired allelic variation during transformation, and had become genetically distinct. MCF-10A-Tr cells formed solid tumors when implanted into the mammary fat pads of Balb/c mice. Data revealed that CSC contained approximately 1.011µg Cd per cigarette equivalent, and Cd (0.0003µg Cd/1×10(7) cells) was also detected in the lysates from MCF-10A cells treated with 25µg/mL CSC. In similar manner to CSC, CdCl2 treatment in MCF-10A cells caused anchorage independent colony growth, higher expression of oncogenic proteins and increased PI3K-AKT-NFκB protein expression. An increase in the expression of PI3K-AKT-NFκB was also noted in the mice xenografts. Interestingly, it was noted that CSC and CdCl2 treatment in MCF-10A cells increased ROS. Collectively, results suggest that heavy metals present in cigarettes of Indian origin may substantially contribute to tumorigenesis by inducing intercellular ROS accumulation and increased expression of PI3K, AKT and NFκB proteins.