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.

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.

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.

Nano formulated Resveratrol inhibits PD-L1 in oral cancer cells by deregulating the association between tumor associated macrophages and cancer associated fibroblasts through IL-6/JAK2/STAT3 signaling axis.

Tumor associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) in the tumor microenvironment secrete several cytokines, which involved in tumor initiation, progression, metastatic outgrowth and angiogenesis. However, the association between TAMs and CAFs in the context of tumor development remain unclear. Here, we studied the relationship between TAMs and CAFs along with the involvement of cytokines in the production of cancer-stem-like-cells (CSCs) in oral cancer cells and explored the potential anticancer effects of Nano-formulated Resveratrol (Res-NP) using an activated macrophage-M1 (AM-M1) and activated fibroblast cells as the model system. IL-6 secretion was found to be enhanced in the conditioned-medium (CM) when AM-M1 cells + CAFs-like cells were cocultured together. CSCs-enriched population was developed after addition of CM of AM-M1 +CAFs in H-357 cells and patient-derived-primary-oral-cancer cells. AM-M1 cells+ CAFs-like cells secreted IL-6 enhanced CSCs growth, proliferation, metastasis, and angiogenesis. IL-6 was found to promote PD-L1 expression in CSCs-enriched cells via JAK2/STAT3 pathway, as evident from the enhanced expression of p-JAK2 and p-STAT3. Nevertheless, Res-NP inhibited CSCs proliferation and reduced the expression of metastatic and angiogenic markers, in ovo blood vascularization, NO production and MMPs expression. Res-NP delinked the association between AM-M1 and CAFs by blocking IL-6 production and also disrupted the potential connection between IL-6 and PD-L1 with considerable decrease in p-JAK2 and p-STAT3 expressions. IL-6 depletion inhibited stemness and angiogenesis in oral CSCs by downregulating PD-L1 via JAK2/STAT3 cascade. Similar observations were also observed in Res-NP treated xenograft mice. Thus, data demonstrate that CSCs growth is dependent on IL-6/PD-L1 axis. Res-NP deregulates the association between AM-M1 and CAFs along-with attenuates carcinogenesis in in vitro, in ovo, ex vivo and in vivo model systems by inhibiting PD-L1 via IL-6/JAK2/STAT3 axis.

Near infrared-responsive quinacrine-gold hybrid nanoparticles deregulate HSP-70/P300-mediated H3K14 acetylation in ER/PR+ breast cancer stem cells.

Aim: This study aimed to determine if quinacrine-gold hybrid nanoparticles (QAuNPs) + near-infrared (NIR) deregulate HSP-70/P300 complex-mediated H3K14 acetylation in estrogen receptor/progesterone receptor (ER/PR+) breast cancer stem cells (CSCs). Materials & methods: Various cells and mouse-based systems were used as models. Results: QAuNP + NIR treatment reduced the nuclear translocation of HSP-70, affected the histone acetyltransferase activity of P300 and specifically decreased H3K14 acetylation in ER/PR+ breast CSCs. Finally, HSP-70 knockdown showed a reduction in P300 histone acetyltransferase activity, decreased H3K14 acetylation and inhibited activation of the TGF-ß gene. Conclusion: This study revealed that QAuNP + NIR irradiation inhibits oncogenic activation of the TGF-ß gene by decreasing H3K14 acetylation mediated through the HSP-70/P300 nuclear complex in ER/PR+ breast CSCs.

Combination of Resveratrol and PARP inhibitor Olaparib efficiently deregulates homologous recombination repair pathway in breast cancer cells through inhibition of TIP60-mediated chromatin relaxation.

Recently, we reported that a combination of a natural, bioactive compound Resveratrol (RES) and a PARP inhibitor Olaparib (OLA) deregulated the homologous recombination (HR) pathway, and enhanced apoptosis in BRCA1-wild-type, HR-proficient breast cancer cells. Upon DNA damage, chromatin relaxation takes place, which allows the DNA repair proteins to access the DNA lesion. But whether chromatin remodeling has any role in RES + OLA-mediated HR inhibition is not known. By using in vitro and ex vivo model systems of breast cancer, we have investigated whether RES + OLA inhibits chromatin relaxation and thereby blocks the HR pathway. It was found that RES + OLA inhibited PARP1 activity, terminated PARP1-BRCA1 interaction, and deregulated the HR pathway only in the chromatin fraction of MCF-7 cells. DR-GFP reporter plasmid-based HR assay demonstrated marked reduction in HR efficiency in I-SceI endonuclease-transfected cells treated with OLA. RES + OLA efficiently trapped PARP1 at the DNA damage site in the chromatin of MCF-7 cells. Unaltered expressions of HR proteins were found in the chromatin of PARP1-silenced MCF-7 cells, which confirmed that RES + OLA-mediated DNA damage response was PARP1-dependent. Histone Acetyltransferase (HAT) activity and histone H4 acetylation assays showed reduction in HAT activity and H4 acetylation in RES + OLA-treated chromatin fraction of cells. Western blot analysis showed that the HAT enzyme TIP60, P400 and acetylated H4 were downregulated after RES + OLA exposure. In the co-immunoprecipitation assay, it was observed that RES + OLA caused abolition of PARP1-TIP60-BRCA1 interaction, which suggested the PARP1-dependent TIP60-BRCA1 association. Unaltered expressions of PAR, BRCA1, P400, and acetylated H4 in the chromatin of TIP60-silenced MCF-7 cells further confirmed the role of TIP60 in PARP1-mediated HR activation in the chromatin. Similar results were obtained in ex vivo patient-derived primary breast cancer cells. Thus, the present study revealed that RES + OLA treatment inhibited PARP1 activity in the chromatin, and blocked TIP60-mediated chromatin relaxation, which, in turn, affected PARP1-dependent TIP60-BRCA1 association, resulting in deregulation of HR pathway in breast cancer cells.

Molecular editing of NSC-666719 enabling discovery of benzodithiazinedioxide-guanidines as anticancer agents.

DNA polymerase ß (Polß) is crucial for the base excision repair (BER) pathway of DNA damage repair and is an attractive target for suppressing tumorigenesis as well as chemotherapeutic intervention of cancer. In this study, a unique strategy of scaffold-hopping-based molecular editing of a bioactive agent NSC-666719 was investigated, which led to the development of new molecular motifs with Polß inhibitory activity. NSC compound and its analogs (two series) were prepared, focusing on pharmacophore-based molecular diversity. Most compounds showed higher activities than the parent NSC-666719 and exhibited effects on apoptosis. The inhibitory activity of Polß was evaluated in both in vitro reconstituted and in vivo intact cell systems. Compound 10e demonstrated significant Polß interaction and inhibition characteristics, including direct, non-covalent, reversible, and comparable binding affinity. The investigated approach is useful, and the discovered novel analogs have a high potential for developing as anticancer therapeutics.

Quinacrine and Curcumin in combination decreased the breast cancer angiogenesis by modulating ABCG2 via VEGF A.

Cancer stem cells (CSCs) cause drug resistance in cancer due to its extensive drug efflux, DNA repair and self-renewal capability. ATP binding cassette subfamily G member 2 (ABCG2) efflux pump afford protection to CSCs in tumors, shielding them from the adverse effects of chemotherapy. Although the role of ABCG2 in cancer progression, invasiveness, recurrence are known but its role in metastasis and angiogenesis are not clear. Here, using in vitro (CSCs enriched side population [SP] cells), ex vivo (patient derived primary cells), in ovo (fertilized egg embryo) and in vivo (patient derived primary tissue mediated xenograft (PDX)) system, we have systematically studied the role of ABCG2 in angiogenesis and the regulation of the process by Curcumin (Cur) and Quinacrine (QC). Cur + QC inhibited the proliferation, invasion, migration and expression of representative markers of metastasis and angiogenesis. Following hypoxia, ABCG2 enriched cells released angiogenic factor vascular endothelial growth factor A (VEGF A) and induced the angiogenesis via PI3K-Akt-eNOS cascade. Cur + QC inhibited the ABCG2 expression and thus reduced the angiogenesis. Interestingly, overexpression of ABCG2 in SP cells and incubation of purified ABCG2 protein in media induced the angiogenesis but knockdown of ABCG2 decreased the vascularization. In agreement with in vitro results, ex vivo data showed similar phenomena. An induction of vascularization was noticed in PDX mice but reduction of vascularization was also observed after treatment of Cur + QC. Thus, data suggested that in hypoxia, ABCG2 enhances the production of angiogenesis factor VEGF A which in turn induced angiogenesis and Cur + QC inhibited the process by inhibiting ABCG2 in breast cancer.

Resveratrol nanoparticle attenuates metastasis and angiogenesis by deregulating inflammatory cytokines through inhibition of CAFs in oral cancer by CXCL-12/IL-6-dependent pathway.

Cancer-associated fibroblasts (CAFs) are one of the highly abundant components in the tumor microenvironment (TME). They secrete several cytokines, which amplified tumor progression, invasion, stemness, metastasis, and angiogenesis. Here, we evaluate the potentiality of cytokines for the formation of cancer stem cells (CSCs) in oral cancer cells niche and investigate the anti-inflammatory and anti-carcinogenic effect of Resveratrol-nanoparticle (Res-NP). We first differentiated quiescent human fibroblasts into CAFs in vitro in response to PDGF-B and TGF-ß stimulation and these CAFs were found to increase CXCL-12 and IL-6 secretion. CSCs-enriched population was created by incubating H-357 cells with CAFs and cytokine-enriched CAFs-conditioned media (CAFs-CM). Likewise, CSCs-populated environment was also generated after incubating CAFs-CM to patient-derived primary oral cancer cells. It was noted that CXCL-12 and IL-6 secreted from CAFs significantly promoted CSCs growth, proliferation, aggressiveness, metastasis, and angiogenesis. However, Res-NP reduced CSCs growth and proliferation by abrogating the secretion of CXCL-12 and IL-6. A significant decrease in the expression of metastatic and angiogenic markers, in ovo blood vascularization, intracellular NO generation, MMPs expression and tube formation was found upon Res-NP treatment. Reduction of representative CSCs and angiogenesis markers were also noted after Res-NP treatment in xenograft mice model. CXCL-12 physically interact with IL-6 and this interaction was diminished after Res-NP treatment. Moreover, the expression of CD133 and VEGF-A were down-regulated either on Res-NP or CXCL-12/IL-6-specific inhibitors treated CSCs-enriched cells. Thus, the data suggest that CSCs growth is CXCL-12 and IL-6 dependent and Res-NP obstruct carcinogenesis and metastasis by inhibiting CXCL-12 and IL-6 production in in vitro, in vivo, in ovo, and ex vivo systems.

Near-infrared enhances antiangiogenic potentiality of quinacrine-gold hybrid nanoparticles in breast cancer stem cells via deregulation of HSP-70/TGF-ß.

Aim: This study aimed to explore the antiangiogenic mechanism of quinacrine-gold hybrid nanoparticle (QAuNP) and near-infrared (NIR) radiation in patient-derived primary breast cancer stem cells. Materials & methods: Various cell-based in ovo angiogenesis and in vivo patient-derived xenograft mouse systems were used as models for the study. Results: The experimental results showed that QAuNP + NIR treatment deregulated the HSP-70/TGF-ß physical interaction in primary breast cancer stem cells. Reduced TGF-ß secretion in the tumor microenvironment inhibited angiogenesis activation in endothelial cells by deregulating the TGF-ß-mediated PI3K/AKT/mTOR cascade. Conclusion: This study revealed that QAuNP + NIR irradiation downregulated HSP-70 expression, inhibited the HSP-70/TGF-ß interaction, reduced the secretion of TGF-ß in the tumor microenvironment and ultimately inhibited TGF-ß-mediated angiogenesis.

This study discovered that the formation of blood vessels in breast cancer is significantly reduced when hybrid nanoparticles and infrared laser therapy are used to treat breast cancer stem cells. The secretory cytokines in the tumor microenvironment primarily responsible for developing blood vessels in the tumor are dramatically reduced by treatment. As a result, the tumor's blood vessel growth is reduced, making it difficult for the cancer cells to get the nutrients and oxygen they need to survive.

Quinacrine inhibits HIF-1α/VEGF-A mediated angiogenesis by disrupting the interaction between cMET and ABCG2 in patient-derived breast cancer stem cells.

BACKGROUND: Breast cancer stem cells (BCSCs) have a critical role in progression of breast cancer by inducing angiogenesis. Several therapeutic strategies have been designed for the treatment of breast cancer by specifically preventing angiogenesis. But there is a dearth of study regarding the treatment procedure which can specifically target and kill the BCSCs and cause lesser harm to healthy cells of the body. A plant-based bioactive compound Quinacrine (QC) specifically kills cancer stem cells (CSCs) without harming healthy cells and also inhibits cancer angiogenesis but the detailed mechanistic study of its anti-CSCs and anti-angiogenic activity is yet to explore. HYPOTHESIS: Earlier report showed that both cMET and ABCG2 play an essential role in cancer angiogenesis. Both are present on the cell surface of CSCs and share an identical ATP-binding domain. Interestingly, QC a plant based and bioactive compound which was found to inhibit the function of CSCs marker cMET and ABCG2. These relevant evidence led us to hypothesize that cMET and ABCG2 may interact with each other and induce the production of angiogenic factors, resulting in activation of cancer angiogenesis and QC might disrupt the interaction between them to stop this phenomena. METHODS: Co-immunoprecipitation assay, immunofluorescence assay, and western blotting were performed by using ex vivo patient-derived breast cancer-stem-cells (PDBCSCs) and human umbilical vein endothelial cells (HUVECs). In silico study was carried out to check the interaction between cMET and ABCG2 in presence or absence of QC. Tube formation assay using HUVECs and in ovo Chorioallantoic membrane (CAM) assay using chick fertilized eggs were performed to monitor angiogenesis. In vivo patient-derived xenograft (PDX) mice model was used to validate in silico and ex vivo results. RESULTS: Data revealed that in a hypoxic tumor microenvironment (TME), cMET and ABCG2 interact with each other and upregulate HIF-1α/VEGF-A axis to induce breast cancer angiogenesis. In silico and ex vivo study showed that QC disrupted the interaction between cMET and ABCG2 to inhibit the angiogenic response in endothelial cells by reducing the secretion of VEGF-A from PDBCSCs within the TME. Knockdown of cMET, ABCG2 or both, significantly downregulated the expression of HIF-1α and reduced the secretion of pro-angiogenic factor VEGF-A in the TME of PDBCSCs. Additionally, when PDBCSCs were treated with QC, similar experimental results were obtained. CONCLUSION: In silico, in ovo, ex vivo and in vivo data confirmed that QC inhibited the HIF-1α/VEGF-A mediated angiogenesis in breast cancer by disrupting the interaction between cMET and ABCG2.

Quinacrine inhibits cMET-mediated metastasis and angiogenesis in breast cancer stem cells.

A trans-membrane receptor tyrosine kinase, cMET, belonging to the MET proto-oncogene family, is responsible for cancer metastasis and angiogenesis. But not much is known about the role of cMET in growth and progression of cancer stem cells (CSCs). Earlier studies have shown that Quinacrine (QC), a bioactive agent, has anti-CSCs activity. Here, the role of QC in deregulation of cMET-mediated metastasis and angiogenesis has been systematically evaluated in vitro in highly metastatic breast CSCs (mBCSCs), ex vivo in patient-derived breast cancer stem cells (PDBCSCs) and in vivo in xenograft mice model systems. Cell proliferation, migration, invasion and representative metastasis markers were upregulated in cMET-overexpressed cells and QC exposure inhibited these processes in both mBCSCs and PDBCSCs. Interestingly, metastasis was significantly inhibited by QC in cMET-overexpressed cells but comparatively lesser significant alteration of the process was noted in cMET-silenced cells. Increase in vascularization (in in ovo CAM assay), and cell-cell tube formation (in HUVECs), and enhanced MMP9 and MMP2 enzymatic activities (in gelatin zymography) were noted after cMET overexpression but these processes got reversed after cMET knockdown or QC treatment in cMET-overexpressed cells. QC inhibited angiogenesis significantly in cMET-overexpressed cells, but lesser significant change was observed in cMET-silenced cells. Reduction in tumor volume and decreased expression of metastatic and angiogenic markers were also noted in xenograft mice after QC treatment. Furthermore, QC inhibited cMET activity by dephosphorylation of its tyrosine residues (Y1234 and Y1356) and downregulation of its downstream cascade. Thus, QC inhibited the cMET-mediated metastasis and angiogenesis in in vitro, in ovo, in vivo and ex vivo model systems. Ligand (HGF) binding leads to receptor dimerization and phosphorylation of tyrosine kinase domain of cMET. This activates the cMET signaling cascade. The representative downstream metastasis and angiogenesis-related proteins get upregulated and induce the metastasis and angiogenesis process. But after the QC treatment, cMET get dephosphorylated and inactivated. As a result, the downstream signaling proteins of cMET along with the other representative metastatic and angiogenic factors get downregulated. These lead to inhibition of cMET-mediated metastasis and angiogenesis. (Created with BioRender.com).

Talazoparib enhances the quinacrine-mediated apoptosis in patient-derived oral mucosa CSCs by inhibiting BER pathway through the modulation of GCN5 and P300.

The presence of cancer stem cells (CSCs) in the tumor microenvironment (TME) is majorly responsible for the development and recurrence of cancer. Earlier reports suggested that upon DNA damage, poly-(ADP-ribose) polymerase-1 (PARP-1) helps in chromatin modulation and DNA repair process, thereby promoting CSC survival. But whether a combination of DNA damaging agents along with PARP inhibitors can modulate chromatin assembly, inhibit DNA repair processes, and subsequently target CSCs is not known. Hence, we have investigated the effect of nontoxic bioactive compound quinacrine (QC) and a potent PARP inhibitor Talazoparib in patient-derived oral mucosa CSCs (OM-CSCs) and in vivo xenograft mice preclinical model systems. Data showed that QC + Talazoparib inhibited the PARP-1-mediated chromatin remodelers' recruitment and deregulated HAT activity of GCN5 (general control nonderepressible-5) and P300 at DNA damage site, thereby preventing the access of repair proteins to the damaged DNA. Additionally, this combination treatment inhibited topoisomerase activity, induced topological stress, and induced apoptosis in OM-CSCs. Similar results were observed in an in vivo xenograft mice model system. Collectively, the data suggested that QC + Talazoparib treatment inhibited BER pathway, induced genomic instability and triggered apoptosis in OM-CSCs through the deregulation of PARP-1-mediated chromatin remodelers (GCN5 and P300) activity. Schematic representation of QC + Talazoparib-induced apoptosis in oral mucosa CSCs. (1) Induction of DNA damage takes place after QC treatment (2) PARP1-mediated PARylation at the site of DNA damage, which recruits multiple chromatin remodelers (3) Acetylation at the histone tails relax the structure of chromatin and recruits the BER pathway proteins at the site of DNA damage. (4) BER pathway activated at the site of DNA damage. (5) CSCs survive after successful repair of DNA damage. (6) Treatment of QC-treated CSCs with PARP inhibitor Talazoparib (7) Inhibition of PARylation results in failure of chromatin remodelers to interact with PARP1. (8) Inhibition of acetylation status leads to chromatin compaction. (9) BER pathway proteins are not recruited at the site of DNA damage, resulting in inhibition of BER pathway and accumulation of unrepaired DNA damage, leading to apoptosis and cell death.

Veliparib (ABT-888), a PARP inhibitor potentiates the cytotoxic activity of 5-fluorouracil by inhibiting MMR pathway through deregulation of MSH6 in colorectal cancer stem cells.

OBJECTIVE: Sensitization of mismatch repair (MMR)-deficient colorectal cancer (CRC) cells by 5-Fluorouracil (5-FU) is well-documented. But not much is known about the treatment of MMR-proficient CRC cancer stem cells (CRC-CSCs). Here, we investigated whether a PARP inhibitor (ABT-888) can enhance the 5-FU-mediated apoptosis in CRC-CSCs through MMR pathway inhibition. METHODS: The anti-cancer action of 5-FU+ABT-888 combination in CRC-CSCs has been studied by using in vitro, ex vivo, and in vivo preclinical model systems. RESULTS: 5-FU caused DNA damage in CRC-CSCs, and ABT-888 enhanced the accumulation of DNA mismatches by downregulating the MMR pathway, triggering S-phase arrest, and finally apoptosis and cell death in 5-FU-pre-treated MMR-proficient-CRC-CSCs at much lower concentrations than their individual treatments. After 5-FU treatment, PARylated-PARP1 activated MMR pathway by interacting with MSH6. But, upon ABT-888 treatment in 5-FU-pre-exposed CSCs, PARylation was inhibited, as a result of which PARP1 could not interact with MSH6, and other MMR proteins were downregulated. The role of MSH6 in PARP1-mediated MMR activation, was confirmed by silencing MSH6 gene, which resulted in MMR pathway shutdown. Similar results were obtained in ex vivo and in vivo model systems. CONCLUSIONS: 5-FU+ABT-888 combination enhanced CRC-CSCs death by increasing DNA damage accumulation and simultaneously inhibiting the MMR pathway in MMR-proficient cells. But this study does not discuss whether the combination treatment will increase the sensitivity of MMR-deficient CSCs, for which further research will be performed in the future.

5-FU is a well-known drug commonly used to treat colorectal cancer and it causes DNA damage inside the cancer cells. The limitation of 5-FU treatment is the development of chemoresistance due to the high DNA repair capacity of cancer stem cells present in the tumor microenvironment. In this study, a novel chemotherapeutic approach has been developed to target colorectal cancer stem cells by using a combination of 5-FU and a PARP1 inhibitor (ABT-888). Here, 5-FU caused DNA damage and ABT-888 enhanced the accumulation of the DNA lesions by inhibiting the MMR repair pathway in 5-FU-pre-treated MMR-proficient-CRC-CSCs. This resulted in S-phase arrest, induction of apoptosis, and finally CSCs death.

Combination of talazoparib and olaparib enhanced the curcumin-mediated apoptosis in oral cancer cells by PARP-1 trapping.

PURPOSE: Inhibition of Poly (ADP-ribose) Polymerases (PARP) results in the blocking of DNA repair cascades that eventually leads to apoptosis and cancer cell death. PARP inhibitors (PARPi) exhibit their actions either by inhibiting PARP-induced PARylation and/or by trapping PARP at the DNA damage site. But, the mechanism of PARPi-mediated induction of cellular toxicity via PARP-trapping is largely unknown. METHODS: The cellular toxicity of PARPi [Talazoparib (BMN) and/or Olaparib (Ola)] was investigated in oral cancer cells and the underlying mechanism was studied by using in vitro, in silico, and in vivo preclinical model systems. RESULTS: The experimental data suggested that induction of DNA damage is imperative for the optimal effectiveness of PARPi. Curcumin (Cur) exhibited maximum DNA damaging capacity in comparison to Resveratrol and 5-Flurouracil. Combination of BMN + Ola induced cell death in Cur pre-treated cells at much lower concentrations than their individual treatments. BMN + Ola treatment deregulated the BER cascade, potentiated PARP-trapping, caused cell cycle arrest and apoptosis in Cur pre-treated cells in a much more effective manner than their individual treatments. In silico data indicated the involvement of different amino acid residues which might play important roles in enhancing the BMN + Ola-mediated PARP-trapping. Moreover, in vivo mice xenograft data also suggested the BMN + Ola-mediated enhancement of apoptotic potentiality of Cur. CONCLUSION: Thus, induction of DNA damage was found to be essential for optimal functioning of PARPi and BMN + Ola combination treatment enhanced the apoptotic potentiality of Cur in cancer cells by enhancing the PARP-trapping activity via modulation of BER cascade.