Targeting signaling pathways in cancer stem cells: A potential approach for developing novel anti-cancer therapeutics.

Cancer stem cells (CSCs) have emerged as prime players in the intricate landscape of cancer development, progression, and resistance to traditional treatments. These unique cellular subpopulations own the remarkable capability of self-renewal and differentiation, giving rise to the diverse cellular makeup of tumors and fostering their recurrence following conventional therapies. In the quest for developing more effective cancer therapeutics, the focus has now shifted toward targeting the signaling pathways that govern CSCs behavior. This chapter underscores the significance of these signaling pathways in CSC biology and their potential as pivotal targets for the development of novel chemotherapy approaches. We delve into several key signaling pathways essential for maintaining the defining characteristics of CSCs, including the Wnt, Hedgehog, Notch, JAK-STAT, NF-κB pathways, among others, shedding light on their potential crosstalk. Furthermore, we highlight the latest advancements in CSC-targeted therapies, spanning from promising preclinical models to ongoing clinical trials. A comprehensive understanding of the intricate molecular aspects of CSC signaling pathways and their manipulation holds the prospective to revolutionize cancer treatment paradigms. This, in turn, could lead to more efficacious and personalized therapies with the ultimate goal of eradicating CSCs and enhancing overall patient outcomes. The exploration of CSC signaling pathways represents a key step towards a brighter future in the battle against cancer.

Nano formulated Resveratrol inhibits metastasis and angiogenesis by reducing inflammatory cytokines in oral cancer cells by targeting tumor associated macrophages.

Tumor associated macrophages in the tumor microenvironment secrete multiple cytokines, which regulate cancer cells growth and invasiveness. We systematically studied the role of cytokines in the induction of cancer stem like cells (CSCs) in oral cancer cells niche and evaluated the mechanism of Resveratrol nanoparticle (Res-Nano) mediated-reduction of CSCs properties in cells. A highly M1-like macrophages-enriched conditioned medium (CM) was generated by treating fixed doses of PMA and LPS in THP-1 cells alone as well as co-cultured of H-357 plus THP-1 cells. These M1-like macrophages increased the production of cytokines (e.g., TNF-α, IL-6, IL-1ß, etc.). A CSCs populated environment was created after addition of cytokine-enriched-CM of co-culture of H-357 and THP-1 cells to cancer cells and cytokine enriched CM of THP-1 cells to patient derived primary oral cancer cells, respectively. After incubation with CM, enhancement of stemness, angiogenic and metastatic properties of both H-357 and primary oral cancer cells were noted. Res-NP decreased the cytokines level in CSCs-enriched cells and reduced the invasion, proliferation and growth of CSCs. Representative metastatic (CD133, ALDH1, CXCR4, etc.) and angiogenic markers (MMPs, iNOS, VEGF-A, etc.) were decreased after Res-NP treatment in CSCs enriched oral cancer cells niche. It also disrupted angiogenesis, depleted nitric oxide production in fertilized chick embryos and reduced the expression of metastatic and angiogenic markers in xenograft mice model system. Thus, this study concluded that CSCs-mediated stemness is a cytokine dependent phenomena and treatment of Res-NP inhibit this process in in vitro, in vivo and ex vivo systems.

PARP inhibitor Veliparib (ABT-888) enhances the anti-angiogenic potentiality of Curcumin through deregulation of NECTIN-4 in oral cancer: Role of nitric oxide (NO).

Concurrent use of DNA damaging agents with PARP inhibitors contribute to the effectiveness of the anticancer therapy. But there is a dearth of reports on the antiangiogenic effects of PARP inhibitors and the suppression of angiogenesis by this drug combination is not yet reported. For the successful development of cancer therapeutics, anti-cancer drugs ought to have anti-angiogenic potentiality along with their DNA damaging abilities. In this current piece of work, we investigated the in vitro and in ovo anti-angiogenic effect of Curcumin and Veliparib (a PARP inhibitor) in oral cancer. Recent evidences suggest an involvement of the NECTIN-4 in cancer angiogenesis and the exact molecular pathway of this involvement remains to be delineated. We observed that the soluble NECTIN-4 secreted from H357 oral cancer cells enhanced the angiogenesis of endothelial cells (HUVECs) and this was inhibited by Curcumin-Veliparib combination. NECTIN-4 enhanced vascularization, induced vasodilation and triggered the angiogenic sprouting via endothelial tip cell filopodia. Data indicated that NECTIN-4 mediated angiogenesis is associated with PI3K-AKT-mediated nitric oxide (NO) formation. A noticeable increase in the NO enhanced epithelial NO level through HIF-1α mediated iNOS activation. We observed that increased NO enhanced the NECTIN-4 mediated eNOS expression and thereby elicited further angiogenesis. Curcumin antagonised the NECTIN-4-induced angiogenesis through inhibition of PI3K-AKT mediated eNOS pathway and Veliparib synergized the effect of Curcumin. Our observations indicate that NO is cardinal in inducing NECTIN-4 mediated angiogenesis in H357 cells. Thus, Curcumin-Veliparib combination suppresses angiogenesis through deregulation of the PI3K-AKT-eNOS pathway downstream to the NECTIN-4.

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.

PARP inhibitor Olaparib Enhances the Apoptotic Potentiality of Curcumin by Increasing the DNA Damage in Oral Cancer Cells through Inhibition of BER Cascade.

Although Olaparib (Ola, a PARP-inhibitor), in combination with other chemotherapeutic agents, was clinically approved to treat prostate cancer, but cytotoxicity, off-target effects of DNA damaging agents limit its applications in clinic. To improve the anti-cancer activity and to study the detailed mechanism of anti-cancer action, here we have used bioactive compound curcumin (Cur) in combination with Ola. Incubation of Ola in Cur pre-treated cells synergistically increased the death of oral cancer cells at much lower concentrations than individual optimum dose and inhibited the topoisomerase activity. Short exposure of Cur caused DNA damage in cells, but more increased DNA damage was noticed when Ola has incubated in Cur pre-treated cells. This combination did not alter the major components of homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways but significantly altered both short patch (SP) and long patch (LP) base excision repair (BER) components in cancer cells. Significant reduction in relative luciferase activity, expression of BER components and PARylation after Cur and Ola treatment confirmed this combination inhibit the BER activity in cells. Reduction of PARylation, decreased expression of BER components, decreased tumor volume and induction of apoptosis were also noticed in Cur + Ola treated Xenograft mice model. The combination treatment of Cur and Ola also helped in recovering the body weight of tumor-bearing mice. Thus, Cur + Ola combination increased the oral cancer cells death by not only causing the DNA damage but also blocking the induction of BER activity.

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.