Cancer-Related Intracellular Signalling Pathways Activated by DOXorubicin/Cyclodextrin-Graphene-Based Nanomaterials

In the last decade, nanotechnological progress has generated new opportunities to improve the safety and efficacy of conventional anticancer therapies. Compared with other carriers, graphene nanoplatforms possess numerous tunable functionalities for the loading of multiple bioactive compounds, although their biocompatibility is still a debated concern. Recently, we have investigated the modulation of genes involved in cancer-associated canonical pathways induced by graphene engineered with cyclodextrins (GCD). Here, we investigated the GCD impact on cells safety, the HEp-2 responsiveness to Doxorubicin (DOX) and the cancer-related intracellular signalling pathways modulated by over time exposure to DOX loaded on GCD (GCD@DOX). Our studies evidenced that both DOX and GCD@DOX induced p53 and p21 signalling resulting in G0/G1 cell cycle arrest. A genotoxic behaviour of DOX was reported via detection of CDK (T14/Y15) activation and reduction of Wee-1 expression. Similarly, we found a cleavage of PARP by DOX within 72 h of exposure. Conversely, GCD@DOX induced a late cleavage of PARP, which could be indicative of less toxic effect due to controlled release of the drug from the GCD nanocarrier. Finally, the induction of the autophagy process supports the potential recycling of DOX with the consequent limitation of its toxic effects. Together, these findings demonstrate that GCD@DOX is a biocompatible drug delivery system able to evade chemoresistance and doxorubicin toxicity.

Novel Small Molecules Capable of Blocking mtRAS-Signaling Pathway

RAS mutants are involved in approximately 30% of all human cancers and have been regarded as undruggable targets owing to relatively smooth protein surface and obscure binding pockets. In our previous study, we have demonstrated that GNF-7, a multi-targeted kinase inhibitor, possesses potent anti-proliferative activity against Ba/F3 cells transformed with NRAS-G12D. Based on our further analysis using Ba/F3 cells transformed with mtRAS, we discovered a series of pyrimido[4,5-d]pyrimidin-2-one analogues as mtRAS-signaling pathway blockers. In addition, our efforts expanded the assessment to cancer cells with mtRAS, which revealed that these substances are also capable of strongly suppressing the proliferation of various cancer cells harboring KRAS-G12D (AsPC-1), KRAS-G12V (SW480, DU-145), KRAS-G12C (H358), KRAS-G13D (MDA-MB-231), KRAS-Q61L (HT-29), and NRAS-Q61L (OCI-AML3). We herein report novel and potent mtRAS-signaling pathway blockers, SIJ1795 and SIJ1772, possessing 2 to 10-fold increased anti-proliferative activities compared to those of GNF-7 on cancer cells harboring mtRAS as well as on Ba/F3 cells transformed with mtRAS. Both SIJ1795 and SIJ1772 attenuate phosphorylation of RAS downstream molecules (AKT and MEK) and induce apoptosis and G0/G1 cell cycle arrest on cancer cells with mtRAS. Moreover, both substances substantially suppress the migration, invasion, and colony formation of cancer cells harboring mtRAS. Taken together, this study led us to identification of SIJ1795 and SIJ1772 capable of strongly inhibiting mtRAS-signaling pathway on cancer cells harboring mtRAS.

eIF5A-Independent Role of DHPS in p21CIP1 and Cell Fate Regulation

Deoxyhypusine synthase (DHPS) catalyzes the first step of hypusination of the elongation translation factor 5A (eIF5A), and these two proteins have an exclusive enzyme–substrate relationship. Here we demonstrate that DHPS has a role independent of eIF5A hypusination in A375 and SK-MEL-28 human melanoma cells, in which the extracellular signal regulated kinase 1/2 (ERK1/2) pathway is deregulated. We found that RNA interference of DHPS induces G0/G1 cell cycle arrest in association with increased p21CIP1 expression in these cells whereas eIF5A knockdown induces cell death without increasing p21CIP1 expression. Interestingly, p21CIP1 knockdown switched DHPS knockdown-induced growth arrest to cell death in these cells, suggesting a specific relation between DHPS and p21CIP1 in determining cell fate. Surprisingly, ectopic expression of DHPS-K329R mutant that cannot hypusinate eIF5A abrogated DHPS knockdown-induced p21CIP1 expression in these cells, suggesting a non-canonical role of DHPS underlying the contrasting effects of DHPS and eIF5A knockdowns. We also show that DHPS knockdown induces p21CIP1 expression in these cells by increasing CDKN1A transcription through TP53 and SP1 in an ERK1/2-dependent manner. These data suggest that DHPS has a role independent of its ability to hypusinate eIF5A in cells, which appears to be important for regulating p21CIP1 expression and cell fate.

Barringtonia asiatica Seed Extract Induces G1 Cell Cycle Arrest in Saccharomyces cerevisiae and Exhibits Cytotoxicity in A2780 Human Ovarian Cancer Cells

Barringtonia asiatica, is traditionally used as a medicinal plant in the Philippines; unfortunately, there is limited evidence supporting its anticancer capability. Thus, our study investigated the cytotoxic effect of B. asiatica seed extract against ovarian carcinoma A2780 (ECACC 93112519) and Saccharomyces cerevisiae as a cancer model organism. Seeds of B. asiatica were freeze-dried and extracted with methanol. The seed extract was investigated for its effect on the cell cycle in S. cerevisiae by utilizing yeast budding experiment and imaging flow cytometry. Its cytotoxicity activity was also tested as well as a live/dead assay was conducted against ovarian cancer cells. Findings revealed the anticancer activity of B. asiatica and its capability to induce G1 cell cycle arrest in S. cerevisiae. Thus, B. asiatica seeds may serve as a potential source of natural compounds towards anticancer drug discovery.

Palbociclib-mediated cell cycle arrest can occur in the absence of the CDK inhibitors p21 and p27

The use of CDK4/6 inhibitors in the treatment of a wide range of cancers is an area of ongoing investigation. Despite their increasing clinical use, there is limited understanding of the determinants of sensitivity and resistance to these drugs. Recent data have cast doubt on how CDK4/6 inhibitors arrest proliferation, provoking renewed interest in the role(s) of CDK4/6 in driving cell proliferation. As the use of CDK4/6 inhibitors in cancer therapies becomes more prominent, an understanding of their effect on the cell cycle becomes more urgent. Here, we investigate the mechanism of action of CDK4/6 inhibitors in promoting cell cycle arrest. Two main models explain how CDK4/6 inhibitors cause G1 cell cycle arrest, which differ in their dependence on the CDK inhibitor proteins p21 and p27. We have used live and fixed single-cell quantitative imaging, with inducible degradation systems, to address the roles of p21 and p27 in the mechanism of action of CDK4/6 inhibitors. We find that CDK4/6 inhibitors can initiate and maintain a cell cycle arrest without p21 or p27. This work clarifies our current understanding of the mechanism of action of CDK4/6 inhibitors and has implications for cancer treatment and patient stratification.