Amal Kamal Abdel-Aziz

Chloroquine synergizes sunitinib cytotoxicity via modulating autophagic, apoptotic and angiogenic machineries

Tyrosine kinases play a pivotal role in oncogenesis. Although tyrosine kinase inhibitors as sunitinib malate are used in cancer therapy, emerging studies report compromised cytotoxicity when used as monotherapy and thus combinations with other anti-cancer agents is recommended. Chloroquine is a clinically available anti-malarial agent which has been shown to exhibit anti-cancer activity. In the current study, we questioned whether chloroquine can modulate sunitinib cytotoxicity. We found that chloroquine synergistically augmented sunitinib cytotoxicity on human breast (MCF-7 and T-47D), cervical (Hela), colorectal (Caco-2 and HCT116), hepatocellular (HepG2), laryngeal (HEp-2) and prostate (PC3) cancer cell lines as indicated by combination and concentration reduction indices. These results were also consistent with that of Ehrlich ascites carcinoma (EAC) Swiss albino mice models as confirmed by tumor volume, weight, histopathological examination and PCNA expression. Sunitinib induced autophagy via upregulating beclin-1 expression which was blocked by chloroquine as evidenced by accumulated SQTSM1/p62 level. Furthermore, chloroquine augmented sunitinib-induced apoptosis by decreasing survivin level and increasing caspase 3 activity. Chloroquine also enhanced the antiangiogenic capacity of sunitinib as indicated by decreased CD34 expression and peritoneal/skin angiogenesis. Sunitinib when combined with chloroquine also increased reactive nitrogen species production via increasing inducible nitric oxide synthase expression and nitric oxide level whilst reduced reactive oxygen species production by increasing GSH level, activities of glutathione peroxidase and catalase and reducing lipid peroxides compared to sunitinib-only treated group. Taken together, these findings suggest that chloroquine enhanced sunitinib cytotoxicity in a synergistic manner via inducing apoptosis while switching off autophagic and angiogenic machineries. Nevertheless, further studies are required to elucidate the efficacy and safety profile of such combination.

Beclin 1 restrains tumorigenesis through Mcl-1 destabilization in an autophagy-independent reciprocal manner

Mcl-1 is a unique Bcl-2 family member that plays crucial roles in apoptosis. Apoptosis-unrelated functions of Mcl-1 are however emerging, further justifying its tight regulation. Here we unravel a novel mechanism of Mcl-1 regulation mediated by the haplo-insufficient tumour suppressor Beclin 1. Beclin 1 negatively modulates Mcl-1 stability in a reciprocal manner whereby depletion of one leads to the stabilization of the other. This co-regulation is independent of autophagy and of their physical interaction. Both Beclin 1 and Mcl-1 are deubiquitinated and thus stabilized by binding to a common deubiquitinase, USP9X. Beclin 1 and Mcl-1 negatively modulate the proteasomal degradation of each other through competitive displacement of USP9X. The analysis of patient-derived melanoma cells and tissue samples shows that the levels of Beclin 1 decrease, while Mcl-1 levels subsequently increase during melanoma progression in a significant inter-dependent manner. The identified inverse co-regulation of Beclin 1 and Mcl-1 represents a mechanism of functional counteraction in cancer.

Discovery of Potent VEGFR-2 Inhibitors based on Furopyrimidine and Thienopyrimidne Scaffolds as Cancer Targeting Agents

Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in cancer angiogenesis. In this study, a series of novel furo[2,3-d]pyrimidine and thieno[2,3-d]pyrimidine based-derivatives were designed and synthesized as VEGFR-2 inhibitors, in accordance to the structure activity relationship (SAR) studies of known type II VEGFR-2 inhibitors. The synthesized compounds were evaluated for their ability to in vitro inhibit VEGFR-2 kinase enzyme. Seven compounds (15b, 16c, 16e, 21a, 21b, 21c and 21e) demonstrated highly potent dose-related VEGFR-2 inhibition with IC50 values in nanomolar range, of which the thieno[2,3-d]pyrimidine based-derivatives (21b, 21c and 21e) exhibited IC50 values of 33.4, 47.0 and 21 nM respectively. Moreover, furo[2,3-d]pyrimidine-based derivative (15b) showed the strongest inhibition of human umbilical vein endothelial cells (HUVEC) proliferation with 99.5% inhibition at 10 μM concentration. Consistent with our in vitro findings, compounds (21b and 21e) orally administered at 5 and 10 mg/kg/day for 8 consecutive days demonstrated potent anticancer activity in Erhlich ascites carcinoma (EAC) solid tumor murine model. Such compounds blunted angiogenesis in EAC as evidenced by reduced percent microvessel via decreasing VEGFR-2 phosphorylation with subsequent induction of apoptotic machinery. Furthermore, Miles vascular permeability assay confirmed their antiangiogenic effects in vivo. Intriguingly, such compounds showed no obvious toxicity.

Dual modulation of MCL-1 and mTOR determines the response to sunitinib

Most patients who initially respond to treatment with the multi–tyrosine kinase inhibitor sunitinib eventually relapse. Therefore, developing a deeper understanding of the contribution of sunitinib’s numerous targets to the clinical response or to resistance is crucial. Here, we have shown that cancer cells respond to clinically relevant doses of sunitinib by enhancing the stability of the antiapoptotic protein MCL-1 and inducing mTORC1 signaling, thus evoking little cytotoxicity. Inhibition of MCL-1 or mTORC1 signaling sensitized cells to clinically relevant doses of sunitinib in vitro and was synergistic with sunitinib in impairing tumor growth in vivo, indicating that these responses are triggered as prosurvival mechanisms that enable cells to tolerate the cytotoxic effects of sunitinib. Furthermore, higher doses of sunitinib were cytotoxic, triggered a decline in MCL-1 levels, and inhibited mTORC1 signaling. Mechanistically, we determined that sunitinib modulates MCL-1 stability by affecting its proteasomal degradation. Dual modulation of MCL-1 stability at different dose ranges of sunitinib was due to differential effects on ERK and GSK3&bgr; activity, and the latter also accounted for dual modulation of mTORC1 activity. Finally, comparison of patient samples prior to and following sunitinib treatment suggested that increases in MCL-1 levels and mTORC1 activity correlate with resistance to sunitinib in patients.

The tyrosine kinase inhibitor, sunitinib malate, induces cognitive impairment in vivo via dysregulating VEGFR signaling, apoptotic and autophagic machineries.

Chemobrain refers to a cluster of cognitive deficits which affects almost 4-75% of chemotherapy-treated cancer patients. Sunitinib, an FDA-approved multityrosine kinase inhibitor, is currently used in treating different types of tumors. Despite being regarded as targeted therapy which blunts sustained angiogenesis in cancer milieu through inhibiting vascular endothelial growth factor receptor 2 (VEGFR2) signaling, the latter has a cardinal role in cognition. Recent clinical reports warned that sunitinib adversely affected memory processing in cancer patients. Nevertheless, the underlying mechanisms have not been investigated yet. Hence, we explored the impact of a clinically relevant dose of sunitinib on memory processing in vivo and questioned the implication of VEGFR2 signaling, autophagy and apoptosis. Strikingly, sunitinib preferentially impaired spatial cognition as evidenced in Morris water maze, T-maze and passive avoidance task. Consistently, sunitinib degenerated cortical and hippocampal neurons as assessed by histopathological examination and toluidine blue staining. Ultrastructural examination also depicted chromatin condensation, mitochondrial damage and accumulated autophagosomes. Digging deeper, central VEGF/VEGFR2/mTOR signaling was robustly suppressed. Besides, sunitinib boosted cortical and hippocampal p53 and executioner caspase-3 and decreased nuclear factor kappa B and Bcl-2 levels promoting apoptotic cell death. It also profoundly impeded neuronal autophagic flux as shown by decreased beclin-1 and Atg5 and increased p62/SQTSM1 levels. To our knowledge, this is the first study to provide molecular insights into sunitinib-induced chemofog where impeded VEGFR2 signaling and autophagic and hyperactivated apoptotic machineries act in neurodegenerative concert. Importantly, our findings shed light on potential therapeutic strategies to be exploited in the management of sunitinib-induced chemobrain.

Modulation of imatinib cytotoxicity by selenite in HCT116 colorectal cancer cells.

Imatinib is a principal therapeutic agent for targeting colorectal tumours. However, mono‐targeting by imatinib does not always achieve complete cancer eradication. Selenite, a well‐known chemopreventive agent, is commonly used in cancer patients. In this study, we aimed to explore whether selenite can modulate imatinib cytotoxicity in colorectal cancer cells. HCT116 cells were treated with different concentrations of imatinib and/or selenite for 24, 48 and 72 hr. Imatinib–selenite interaction was analysed using isobologram equation. As indicators of apoptosis, DNA fragmentation, caspase‐3 activity, Bcl‐2 expression were explored. Autophagic machinery was also checked by visualizing acidic vesicular organelles and measuring Beclin‐1 expression. Furthermore, reactive oxygen and nitrogen species were also examined. This study demonstrated that selenite synergistically augmented imatinib cytotoxicity in HCT116 cells as demonstrated by combination and dose reduction indices. Supranutritional dose of selenite when combined with imatinib induced apoptotic machinery by decreasing Bcl‐2 expression, increasing caspase‐3 activity and subsequently fragmenting DNA and blunted cytoprotective autophagy by decreasing Beclin‐1 expression and autophagosomes formation. Moreover, their combination induced cell cycle S‐phase block, increased total thiol content and reduced nitric oxide levels. In conclusion, selenite synergizes imatinib cytotoxicity through multi‐barrelled molecular targeting, providing a novel therapeutic approach for colorectal cancer.

From Resistance to Sensitivity: Insights and Implications of Biphasic Modulation of Autophagy by Sunitinib

Sunitinib, a multityrosine kinase inhibitor, is currently the standard first-line therapy in metastatic renal cell carcinoma (mRCC) and is also used in treating patients with pancreatic neuroendocrine and imatinib-resistant gastrointestinal stromal tumors (GIST). Nevertheless, most patients eventually relapse secondary to intrinsic or acquired sunitinib resistance. Autophagy has been reported to contribute to both chemo-sensitivity and -resistance. However, over the last few years, controversial regulatory effects of sunitinib on autophagy have been reported. Since gaining insights into the underlying molecular insights and clinical implications is indispensible for achieving optimum therapeutic response, this minireview article sheds light on the role of a network of prosurvival signaling pathways recently identified as key mediators of sunitinib resistance with established and emerging functions as autophagy regulators. Furthermore, we underscore putative prognostic biomarkers of sunitinib responsiveness that could guide clinicians toward patient stratification and more individualized therapy. Importantly, innovative therapeutic strategies/approaches to overcome sunitinib resistance both evaluated in preclinical studies and perspective clinical trials are discussed which could ultimately be translated to better clinical outcome.

Comparative study of anti-VEGF Ranibizumab and interleukin-6 receptor antagonist tocilizumab in adjuvant-induced arthritis.

&NA; Although the precise etiology of Rheumatoid arthritis (RA) remains obscure, heightened immune response is thought to play a vital role in provoking joint inflammation and bone erosion. This study aims at comparatively evaluating the effects of two monoclonal antibodies Ranibizumab (RANI) as anti‐VEGF antibody and Tocilizumab (TCZ) as interleukin‐6 receptor (IL‐6R) antagonist, against adjuvant induced arthritis in rats. CFA‐induced arthritic rats were treated for three consecutive weeks with Methotrexate (MTX), TCZ and RANI monotherapy. Clinical assessment of RA, bone erosion, inflammatory, angiogenic and apoptotic markers were determined to assess the anti‐arthritic effect. Liver enzymes and histopathological examination of liver and spleen were assessed to evaluate the toxicity profile of the tested therapeutic agents. MTX, TCZ and RANI monotherapy significantly enhanced the anti‐arthritic parameters in comparison with the Complete Freunds Adjuvant (CFA)‐induced arthritic rats through significant reduction of ankle and paw swelling. Also, they significantly reduced inflammatory, angiogenic and apoptotic markers. Importantly, Ranibizumab showed better effect than the standard anti‐rheumatic drugs Methotrexate (MTX) or Tocilizumab (TCZ) in bone protection and cartilage health; hence proves to be a promising new therapeutic agent for RA. Graphical abstract RA development starts with triggering immune cells such as Macrophages and Neutrophils. These cells produced different types of cytokines such as IL‐6 and TNF‐&agr;. IL‐6 with TNF‐&agr; participate in T‐cells differentiation into Th‐17 that produce IL‐17. IL‐17 plays a synergistic role with other cytokines in immune cells differentiation. Il‐6 is also involved in differentiation of B‐cells into plasma cells. Furthermore, IL‐6 and IL‐1b enhance synovocytes proliferation that lead to angiogenesis, apoptosis and cartilage destruction and bone erosion through production of different cytokines, markers and enzymes such as VEGF, TGF&bgr;, COX‐II, RANKL and MMPs. Figure. No caption available. HighlightsRANI significantly reduced ankle and paw swelling in CFA‐induced arthritic rats.Anti‐inflammatory of RANI was confirmed beside its anti‐angiogenic effect.RANI also produced anti‐apoptotic and bone protective effects.RANI modulated the interplay between different arthritic machineries.RANI showed better bone protective effect than MTX or TCZ monotherapy.

Corrigendum: From Resistance to Sensitivity: Insights and Implications of Biphasic Modulation of Autophagy by Sunitinib.

[This corrects the article on p. 718 in vol. 8, PMID: 29066973.].

Surmounting the resistance against EGFR inhibitors through the development of thieno[2,3-d]pyrimidine-based dual EGFR/HER2 inhibitors

In light of the emergence of resistance against the currently available EGFR inhibitors, our study focuses on tackling this problem through the development of dual EGFR/HER2 inhibitors with improved enzymatic affinities. Guided by the binding mode of the marketed dual EGFR/HER2 inhibitor, Lapatinib, we proposed the design of dual EGFR/HER2 inhibitors based on the 6-phenylthieno[2,3-d]pyrimidine as a core scaffold and hinge binder. After two cycles of screening aiming to identify the optimum aniline headgroup and solubilizing group, we eventually identified 27b as a dual EGFR/HER2 inhibitor with IC50 values of 91.7 nM and 1.2 μM, respectively. Notably, 27b dramatically reduced the viability of various patient-derived cancer cells preferentially overexpressing EGFR/HER2 (A431, MDA-MBA-361 and SKBr3 with IC50 values of 1.45, 3.5 and 4.83 μM, respectively). Additionally, 27b efficiently thwarted the proliferation of lapatinib-resistant human non-small lung carcinoma (NCI-H1975) cells, harboring T790 M mutation, with IC50 of 4.2 μM. Consistently, 27b significantly blocked EGF-induced EGFR activation and inactivated its downstream AKT/mTOR/S6 signalling pathway triggering apoptotic cell death in NCI-H1975 cells. The present study presents a promising candidate for further design and development of novel EGFR/HER2 inhibitors capable of overcoming EGFR TKIs resistance.