Nanoparticle-mediated cancer cell therapy: basic science to clinical applications.

Every sixth person in the world dies due to cancer, making it the second leading severe cause of death after cardiovascular diseases. According to WHO, cancer claimed nearly 10 million deaths in 2020. The most common types of cancers reported have been breast (lung, colon and rectum, prostate cases), skin (non-melanoma) and stomach. In addition to surgery, the most widely used traditional types of anti-cancer treatment are radio- and chemotherapy. However, these do not distinguish between normal and malignant cells. Additional treatment methods have evolved over time for early detection and targeted therapy of cancer. However, each method has its limitations and the associated treatment costs are quite high with adverse effects on the quality of life of patients. Use of individual atoms or a cluster of atoms (nanoparticles) can cause a paradigm shift by virtue of providing point of sight sensing and diagnosis of cancer. Nanoparticles (1-100 nm in size) are 1000 times smaller in size than the human cell and endowed with safer relocation capability to attack mechanically and chemically at a precise location which is one avenue that can be used to destroy cancer cells precisely. This review summarises the extant understanding and the work done in this area to pave the way for physicians to accelerate the use of hybrid mode of treatments by leveraging the use of various nanoparticles.

Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.

We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT.

Clinical resistance associated with a novel MAP2K1 mutation in a patient with Langerhans cell histiocytosis.

Patients with Langerhans cell histiocytosis (LCH) harbor BRAF V600E and activating mutations of MAP2K1/MEK1 in 50% and 25% of cases, respectively. We evaluated a patient with treatment-refractory LCH for mutations in the RAS-RAF-MEK-ERK pathway and identified a novel mutation in the MAP2K1 gene resulting in a p.L98_K104 > Q deletion and predicted to be auto-activating. During treatment with the MEK inhibitor trametinib, the patient's disease showed significant progression. In vitro characterization of the MAP2K1 p.L98_K104 > Q deletion confirmed its effect on cellular activation of the ERK pathway and drug resistance.

Downregulation of IGF-1 receptor occurs after hepatic linage commitment during hepatocyte differentiation from human embryonic stem cells.

The insulin-like growth factor 1 receptor (IGF-1R) has been suggested to be involved in hepatocyte differentiation. Human hepatocyte cancer cells and stem cells are known to express IGF-1R whereas normal hepatocytes do not. In the present study we optimized a differentiation protocol and verified the different stages by established markers. The expression levels of IGF-1R and major downstream signaling proteins during differentiation from human embryonic stem cells (hESC) to mature hepatocytes were investigated. We could only demonstrate a minor decrease in IGF-1R expression during endodermal differentiation compared to hESC, but declined substantially (>50%) after hepatic lineage commitment during the hepatocyte specification and maturation stages. This downregulation was paralleled by an upregulation of ERK 1/2, AKT and insulin substrate-1. Neither inhibition nor activation of IGF-1R had any essential effect on endoderm differentiation of human embryonic stem cells. Therefore, our data suggest that IGF-1R downregulation may have a regulatory impact after initiation of hepatic lineage commitment.

Identifying lifestyle factors associated to co-morbidity of obesity and psychiatric disorders, a pilot study.

Obesity and psychiatric disorders are linked through a bidirectional association. Obesity rates have tripled globally in the past decades, and it is predicted that by 2025, one billion people will be affected by obesity, often with a co-morbidity such as depression. While this co-morbidity seems to be a global health issue, lifestyle factors associated to it differ between countries and are often attributed to more than one factor. Prior obesity studies were performed in Western populations; this is the first study that investigates lifestyle factors relating to obesity and mental health of the diverse population in Qatar, a country that has witnessed tremendous lifestyle change in a short time. In this pilot study, we surveyed 379 respondents to assess and compare the lifestyles of Qatar residents to the global population. However due to the high proportion of responses from the United Kingdom (UK) residents, we have made comparisons between Qatar residents and UK residents. We used chi-square analysis, spearman rank correlation and logistic regression to compare the lifestyle factors of individuals suffering from both increased BMI and mental health conditions. The types of food consumed, stress, exercise frequency and duration, alcohol and tobacco consumption, and sleep duration, were explored and results argue that different lifestyle factors can contribute to the same health condition, suggesting different mechanisms involved. We found that both groups reported similar sleep durations (p = 0.800), but that perception of sleep (p = 0.011), consumption of alcohol (p = 0.001), consumption of takeaway food (p = 0.007), and physical activity significantly varied between the groups (p = 0.0001). The study examined the predictors of comorbidity in Qatar as well as UK populations using multivariate logistic regression analysis. The result of the study showed no statistical association between comorbidity and the predictors drinking habit, smoking, physical activity, vegetable consumption, eat outs, and sleep perception for the Qatar population, and for the combined population. This study, however showed a significant association (p = 0.033) between sleep perception and comorbidity for the UK population. We conclude that further analysis is needed to understand the relationship between specific lifestyle factors and multimorbidity in each country.

IL-7 promotes T cell proliferation through destabilization of p27Kip1.

Interleukin (IL)-7 is required for survival and homeostatic proliferation of T lymphocytes. The survival effect of IL-7 is primarily through regulation of Bcl-2 family members; however, the proliferative mechanism is unclear. It has not been determined whether the IL-7 receptor actually delivers a proliferative signal or whether, by promoting survival, proliferation results from signals other than the IL-7 receptor. We show that in an IL-7-dependent T cell line, cells protected from apoptosis nevertheless underwent cell cycle arrest after IL-7 withdrawal. This arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27Kip1 through a posttranslational mechanism. Overexpression of p27Kip1 induced G1 arrest in the presence of IL-7, whereas knockdown of p27Kip1 by small interfering RNA promoted S phase entry after IL-7 withdrawal. CD4 or CD8 T cells transferred into IL-7-deficient hosts underwent G1 arrest, whereas 27Kip1-deficient T cells underwent proliferation. We observed that IL-7 withdrawal activated protein kinase C (PKC)theta and that inhibition of PKCtheta with a pharmacological inhibitor completely blocked the rise of p27Kip1 and rescued cells from G1 arrest. The conventional pathway to breakdown of p27Kip1 is mediated by S phase kinase-associated protein 2; however, our evidence suggests that PKCtheta acts via a distinct, unknown pathway inducing G1 arrest after IL-7 withdrawal from T cells. Hence, IL-7 maintains T cell proliferation through a novel pathway of p27Kip1 regulation.

Cdc2-cyclin E complexes regulate the G1/S phase transition.

The cyclin-dependent kinase inhibitor p27(Kip1) is known as a negative regulator of cell-cycle progression and as a tumour suppressor. Cdk2 is the main target of p27 (refs 2, 3) and therefore we hypothesized that loss of Cdk2 activity should modify the p27(-/-) mouse phenotype. Here, we show that although p27(-/-) Cdk2(-/-) mice developed ovary tumours and tumours in the anterior lobe of the pituitary, we failed to detect any functional complementation in p27(-/-) Cdk2(-/-) double-knockout mice, indicating a parallel pathway regulated by p27. We observed elevated levels of S phase and mitosis in tissues of p27(-/-) Cdk2(-/-) mice concomitantly with elevated Cdc2 activity in p27(-/-) Cdk2(-/-) extracts. p27 binds to Cdc2, cyclin B1, cyclin A2, or suc1 complexes in wild-type and Cdk2(-/-) extracts. In addition, cyclin E binds to and activates Cdc2. Our in vivo results provide strong evidence that Cdc2 may compensate the loss of Cdk2 function.

Upregulation of the insulin receptor and type I insulin-like growth factor receptor are early events in hepatocarcinogenesis.

The molecular mechanisms underlying the development of hepatocellular carcinoma (HCC) are not yet fully understood. Preneoplastic foci of altered hepatocytes regularly precede HCC in various species. The predominant earliest type of foci of altered hepatocytes, the glycogen storage focus (GSF), shows an excess of glycogen (glycogenosis) in the cytoplasm. During progression from GSF to HCC, the stored glycogen is gradually reduced, resulting in complete loss in basophilic HCC. We have previously shown that in N-nitrosomorpholine-induced hepatocarcinogenesis, insulin receptor substrate (IRS-1) is strongly expressed in GSF and reduced during progression to HCC, thus correlating with the glycogen content. In the present study, we observed increased levels of insulin receptor, IGF-I receptor (IGF-IR), IRS-2, and mitogen-activated kinase/extracellular regulated kinase-1 in GSF, following the same pattern of expression as IRS-1. We conclude that the abundance of IRS-1, IRS-2, and mitogen-activated kinase/extracellular regulated kinase-1 coincides with a concerted upregulation of both IR and IGF-IR induced by the hepatocarcinogen. Our data suggest that in early hepatocellular preneoplasia, the upregulation of IR elicits glycogenosis through IRS-1 and/or IRS-2, whereas the increased level of the IGF-IR may lead to the increased cell proliferation previously reported in GSF. Therefore, the concerted upregulation of both IR and IGF-IR may represent initial events in hepatocarcinogenesis.

Highlights on selected growth factors and their receptors as promising anticancer drug targets.

Growth factor receptors (GFRs) and receptor tyrosine kinases (RTK) are groups of proteins mediating a plethora of physiological processes, including cell growth, proliferation, survival, differentiation and migration. Under certain circumstances, expression of GFRs and subsequently their downstream kinase signaling are deregulated by genetic, epigenetic, and somatic changes leading to uncontrolled cell division in many human diseases, most notably cancer. Cancer cells rely on growth factors to sustain the increasing need to cell division and metabolic reprogramming through cancer-associated activating mutations of their receptors (i.e., GFRs). In this review, we highlight the recent advances of selected GFRs and their ligands (growth factors) in cancer with emphasis on structural and functional differences. We also interrogate how overexpression and/or hyperactivation of GFRs contribute to cancer initiation, development, progression, and resistance to conventional chemo- and radiotherapies. Novel approaches are being developed as anticancer agents to target growth factor receptors and their signaling pathways in different cancers. Here, we illustrate how the current knowledge of GFRs biology, and their ligands lead to development of targeted therapies to inhibit and/or block the activity of growth factors, GFRs and downstream kinases to treat diseases such as cancer.

Exopolysaccharide-peptide complex from oyster mushroom (Pleurotus ostreatus) protects against hepatotoxicity in rats.

Liver damage involves oxidative stress and a progression from chronic hepatitis to hepatocellular carcinoma (HCC). The increased incidence of liver disease in Egypt and other countries in the last decade, coupled with poor prognosis, justify the critical need to introduce alternative chemopreventive agents that may protect against liver damage. The aim of this study was to evaluate the efficacy of exopolysaccharide-peptide (PSP) complex extracted from Pleurotus ostreatus as a hepatoprotective agent against diethylnitrosamine (DEN)/carbon tetrachloride (CCL4)-induced hepatocellular damage in rats. The levels of liver injury markers (ALT, AST and ALP) were substantially increased following DEN/CCl4 treatment. DEN/CCl4 - induced oxidative stress was confirmed by elevated levels of lipid peroxidation and decreased levels of superoxide dismutase, glutathione-S-transferase, and reduced glutathione. PSP reversed these alterations in the liver and serum, and provided protection evidenced by reversal of histopathological changes in the liver. The present study demonstrated that PSP extract from P. ostreatus exhibited hepatoprotective and antioxidant effects against DEN/CCl4-induced hepatocellular damage in rats. Given the high prevalence of HCV-related liver damage in Egypt, our results suggest further clinical evaluation of P. ostreatus extracts and their potential hepatoprotective effects in patients with liver disease.

PRKAR1A inactivation leads to increased proliferation and decreased apoptosis in human B lymphocytes.

The multiple neoplasia syndrome Carney complex (CNC) is caused by heterozygote mutations in the gene, which codes for the RIalpha regulatory subunit (PRKAR1A) of protein kinase A. Inactivation of PRKAR1A and the additional loss of the normal allele lead to tumors in CNC patients and increased cyclic AMP signaling in their cells, but the oncogenetic mechanisms in affected tissues remain unknown. Previous studies suggested that PRKAR1A down-regulation may lead to increased mitogen-activated protein kinase (MAPK) signaling. Here, we show that, in lymphocytes with PRKAR1A-inactivating mutations, there is increased extracellular signal-regulated kinase (ERK) 1/2 and B-raf phosphorylation and MAPK/ERK kinase 1/2 and c-Myc activation, whereas c-Raf-1 is inhibited. These changes are accompanied by increased cell cycle rates and decreased apoptosis that result in an overall net gain in proliferation and survival. In conclusion, inactivation of PRKAR1A leads to widespread changes in molecular pathways that control cell cycle and apoptosis. This is the first study to show that human cells with partially inactivated RIalpha levels have increased proliferation and survival, suggesting that loss of the normal allele in these cells is not necessary for these changes to occur.

Cdk2 knockout mice are viable.

BACKGROUND: Cyclin-dependent kinases (Cdks) and their cyclin regulatory subunits control cell growth and division. Cdk2/cyclin E complexes are thought to be required because they phosphorylate the retinoblastoma protein and drive cells through the G1/S transition into the S phase of the cell cycle. In addition, Cdk2 associates with cyclin A, which itself is essential for cell proliferation during early embryonic development. RESULTS: In order to study the functions of Cdk2 in vivo, we generated Cdk2 knockout mice. Surprisingly, these mice are viable, and therefore Cdk2 is not an essential gene in the mouse. However, Cdk2 is required for germ cell development; both male and female Cdk2(-/-) mice are sterile. Immunoprecipitates of cyclin E1 complexes from Cdk2(-/-) spleen extracts displayed no activity toward histone H1. Cyclin A2 complexes were active in primary mouse embryonic fibroblasts (MEFs), embryo extracts and in spleen extracts from young animals. In contrast, there was little cyclin A2 kinase activity in immortalized MEFs and spleen extracts from adult animals. Cdk2(-/-) MEFs proliferate but enter delayed into S phase. Ectopic expression of Cdk2 in Cdk2(-/-) MEFs rescued the delayed entry into S phase. CONCLUSIONS: Although Cdk2 is not an essential gene in the mouse, it is required for germ cell development and meiosis. Loss of Cdk2 affects the timing of S phase, suggesting that Cdk2 is involved in regulating progression through the mitotic cell cycle.

Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells.

BACKGROUND: Children with Down syndrome (DS) have increased risk for developing AML (DS-AMKL), and they usually experience severe therapy-related toxicities compared to non DS-AMKL. Refractory/relapsed disease has very poor outcome, and patients would benefit from novel, less toxic, therapeutic strategies that overcome resistance. Relapse/resistance are linked to cancer stem cells with high aldehyde dehydrogenase (ALDH) activity. The purpose of the present work was to study less toxic alternative therapeutic agents for relapsed/refractory DS-AMKL. METHODS: Fourteen AML cell lines including the DS-AMKL CMY and CMK from relapsed/refractory AML were used. Cytarabine (Ara-C), bortezomib (BTZ), disulfiram/copper (DSF/Cu2+) were evaluated for cytotoxicity, depletion of ALDH-positive cells, and resistance. BTZ-resistant CMY and CMK variants were generated by continuous BTZ treatment. Cell viability was assessed using CellTiter-Glo®, ALDH activity by ALDELUORTM, and proteasome inhibition by western blot of ubiquitinated proteins and the Proteasome-Glo™ Chymotrypsin-Like (CT-like) assay, apoptosis by Annexin V Fluos/Propidium iodide staining, and mutations were detected using PCR, cloning and sequencing. RESULTS: Ara-C-resistant AML cell lines were sensitive to BTZ and DSF/Cu2+. The Ara-C-resistant DS-AMKL CMY cells had a high percentage of ALDHbright "stem-like" populations that may underlie Ara-C resistance. One percent of these cells were still resistant to BTZ but sensitive to DSF/Cu2+. To understand the mechanism of BTZ resistance, BTZ resistant (CMY-BR) and (CMK-BR) were generated. A novel mutation PSMB5 Q62P underlied BTZ resistance, and was associated with an overexpression of the ß5 proteasome subunit. BTZ-resistance conferred increased resistance to Ara-C due to G1 arrest in the CMY-BR cells, which protected the cells from S-phase damage by Ara-C. CMY-BR and CMK-BR cells were cross-resistant to CFZ and MG-132 but sensitive to DSF/Cu2+. In this setting, DSF/Cu2+ induced apoptosis and proteasome inhibition independent of CT-like activity inhibition. CONCLUSIONS: We provide evidence that DSF/Cu2+ overcomes Ara-C and BTZ resistance in cell lines from DS-AMKL patients. A novel mutation underlying BTZ resistance was detected that may identify BTZ-resistant patients, who may not benefit from treatment with CFZ or Ara-C, but may be responsive to DSF/Cu2+. Our findings support the clinical development of DSF/Cu2+ as a less toxic efficacious treatment approach in patients with relapsed/refractory DS-AMKL.

Cdk2 as a master of S phase entry: fact or fake?

It has long been believed that Cdk2 and its activator cyclin E play essential roles in the progression of the mitotic cell cycle. However, recent studies using knockout mouse models revealed that neither Cdk2 nor cyclin E are essential in vivo. The purpose of this Perspective is to compare both Cdk2 and cyclin E knockout mice models and to discuss potential mechanisms driving the cell cycle in the absence of Cdk2 or cyclin E. Particular emphasis is placed on possible non-catalytic roles of cyclin E, the expression and activity of the second cyclin binding partner of Cdk2, cyclin A, as well as on the expression and degradation of the Cdk2 inhibitor p27Kip1 in the absence of Cdk2.

Targeting cell cycle regulators in hematologic malignancies.

Hematologic malignancies represent the fourth most frequently diagnosed cancer in economically developed countries. In hematologic malignancies normal hematopoiesis is interrupted by uncontrolled growth of a genetically altered stem or progenitor cell (HSPC) that maintains its ability of self-renewal. Cyclin-dependent kinases (CDKs) not only regulate the mammalian cell cycle, but also influence other vital cellular processes, such as stem cell renewal, differentiation, transcription, epigenetic regulation, apoptosis, and DNA repair. Chromosomal translocations, amplification, overexpression and altered CDK activities have been described in different types of human cancer, which have made them attractive targets for pharmacological inhibition. Mouse models deficient for one or more CDKs have significantly contributed to our current understanding of the physiological functions of CDKs, as well as their roles in human cancer. The present review focuses on selected cell cycle kinases with recent emerging key functions in hematopoiesis and in hematopoietic malignancies, such as CDK6 and its role in MLL-rearranged leukemia and acute lymphocytic leukemia, CDK1 and its regulator WEE-1 in acute myeloid leukemia (AML), and cyclin C/CDK8/CDK19 complexes in T-cell acute lymphocytic leukemia. The knowledge gained from gene knockout experiments in mice of these kinases is also summarized. An overview of compounds targeting these kinases, which are currently in clinical development in various solid tumors and hematopoietic malignances, is presented. These include the CDK4/CDK6 inhibitors (palbociclib, LEE011, LY2835219), pan-CDK inhibitors that target CDK1 (dinaciclib, flavopiridol, AT7519, TG02, P276-00, terampeprocol and RGB 286638) as well as the WEE-1 kinase inhibitor, MK-1775. The advantage of combination therapy of cell cycle inhibitors with conventional chemotherapeutic agents used in the treatment of AML, such as cytarabine, is discussed.

Protein phosphatase inhibitor-1 mRNA expression correlates with neoplastic transformation of epithelial liver cells and progression of hepatocellular carcinomas.

Protein phosphatase inhibitor-1 plays an important role in the regulation of glycogen metabolism through inhibition of protein phosphatase-1 activity, and it has been implicated in the regulation of cell growth. Using real-time quantitative RT-PCR, we studied the mRNA expression of inhibitor-1 in hepatocellular carcinomas induced in rats by oral administration of N-nitrosomorpholine, and in a non-tumorigenic liver cell line (C1I), that stores glycogen in excess during early passages. In late passages, glycogen is gradually lost concomitant with cell transformation. Our in vitro model included a tumorigenic subline of C1I cells that was obtained by chemically-induced neoplastic transformation using N-methyl-N'-nitro-N-nitrosoguanidine (C1Ict), and does not store glycogen, as well as Morris hepatoma 3924A (MH3924A) cells. We found that in hepatocellular carcinomas, in the late glycogen-poor passages (C1I(late)), and in the tumorigenic subline (C1Ict) of C1I cells, and in MH3924A cells the mRNA expression of inhibitor-1 is significantly increased. This increase in expression varied from 15 to 290-fold of that observed in normal liver. In contrast, in the early glycogen-storing passage of C1I cells (C1I(early)) the level of inhibitor-1 mRNA was found to be slightly less than that of normal liver. Inhibitor-1 mRNA levels correlated with the degree of differentiation of HCCs. These results indicate that the expression of inhibitor-1 mRNA is tightly linked to tumor progression and to the process of liver cell transformation in vitro and is inversely correlated with the glycogen content of the cell.

Picropodophyllin causes mitotic arrest and catastrophe by depolymerizing microtubules via insulin-like growth factor-1 receptor-independent mechanism.

Picropodophyllin (PPP) is an anticancer drug undergoing clinical development in NSCLC. PPP has been shown to suppress IGF-1R signaling and to induce a G2/M cell cycle phase arrest but the exact mechanisms remain to be elucidated. The present study identified an IGF-1-independent mechanism of PPP leading to pro-metaphase arrest. The mitotic block was induced in human cancer cell lines and in an A549 xenograft mouse but did not occur in normal hepatocytes/mouse tissues. Cell cycle arrest by PPP occurred in vitro and in vivo accompanied by prominent CDK1 activation, and was IGF-1R-independent since it occurred also in IGF-1R-depleted and null cells. The tumor cells were not arrested in G2/M but in mitosis. Centrosome separation was prevented during mitotic entry, resulting in a monopolar mitotic spindle with subsequent prometaphase-arrest, independent of Plk1/Aurora A or Eg5, and leading to cell features of mitotic catastrophe. PPP also increased soluble tubulin and decreased spindle-associated tubulin within minutes, indicating that it interfered with microtubule dynamics. These results provide a novel IGF-1R-independent mechanism of antitumor effects of PPP.

ß-Glucans and their applications in cancer therapy: focus on human studies.

ß-glucans belong to a group of polysaccharides located in the cell wall of bacteria, fungi including mushrooms, as well as cereals such as barley and oats. All ß-glucans are glucose polymers linked together by a (ß 1-3) linear ß-glycosidic chain core and they differ by their length and branching structures. They are considered biological response modifiers with immunomodulatory and health beneficial effects including anticancer properties. Few studies using purified ß- glucans were performed, but their anticancer potential was demonstrated mainly through studies using extracts from mushrooms, yeast or other sources which contain ß-glucan as a key component. Their anticancer effects were demonstrated mainly in in vitro and in vivo experimental systems but fewer studies from human populations are available. ß-glucans have been used as adjuvant therapy in clinical trials, mainly in the Far East, with a positive effect on patients'survival and quality of life. The mechanism of action is suggested to be through its stimulation of the immune system. This review focuses on human studies; clinical trials and epidemiological data assessing the efficacy and safety of mushroom-derived ß- glucans in cancer treatment and prevention. The potential direct effects of ß-glucans on cancer cells are also described.

CDK2 knockdown enhances head and neck cancer cell radiosensitivity.

PURPOSE: Cyclin-dependent kinase 2 (CDK2) is critically involved in cell cycling and has been proposed as a potential cancer target. It remains largely elusive whether CDK2 targeting alters the tumor cell radiosensitivity. MATERIALS AND METHODS: CDK2(-/-) and wild type (WT) mouse embryonic fibroblasts (MEF) as well as six human head and neck squamous cell carcinoma (HNSCC) cell lines (SAS, FaDu, Cal-33, HSC-4, UTSCC-5, UTSCC-8) were used. Upon CDK2 knockdown using small interfering technology, colony formation, DNA double-strand breaks (DSB), cell cycle distribution and expression and phosphorylation of major proteins regulating cell cycle and DNA damage repair were examined. RESULTS: CDK2(-/-) MEF and CDK2 HNSCC knockdown cell cultures were more radiosensitive than the corresponding controls. Repair of DSB was attenuated under CDK2 knockout or knockdown. In contrast to data in MEF, combined CDK2 knockdown with irradiation showed no cell cycling alterations in SAS and FaDu cultures. Importantly, CDK2 knockdown failed to radiosensitize SAS and FaDu when cultured in a more physiological three-dimensional (3D) extracellular matrix environment. CONCLUSIONS: Our findings suggest that targeting of CDK2 radiosensitizes HNSCC cells growing as monolayer. Additional studies performed under more physiological conditions are warranted to clarify the potential of CDK2 as target in radiotherapy.

Multiple antitumor effects of picropodophyllin in colon carcinoma cell lines: clinical implications.

Although colorectal cancer can be successfully treated by conventional strategies such as chemo/radiotherapy and surgery, a substantial number of cases, in particular those with liver metastases, remain incurable. Therefore, novel treatment approaches are warranted. The IGF-1R and its ligands, mainly IGF-1 and IGF-2, have been suggested to play pivotal roles in proliferation, survival and migration of adenocarcinoma cells of the colon/rectum. Therefore, interference with IGF-1R-mediated signaling may represent a therapeutic option for this malignancy. In this study, semi-quantitative RT-PCR analyses of 48 paired, colorectal cancer patient samples showed significant overexpression of tumor IGF-1R and IGF-2 mRNA. There was also an overexpression of MMP-7, which was significantly correlated with histopathological parameters. Based on these findings, the effect of the IGF-1R-inhibitory cyclolignan picropodophyllin (PPP) was assessed in the four colon carcinoma cell lines HT-29, HCT-116, DLD-1 and CaCO-2. PPP strongly and dose-dependently inhibited proliferation and migration in all cell lines. However, when exposed to 0.5 µM PPP, only HT-29 showed a net decrease of viable cells as compared with the cell number at the beginning of the experiment, a finding that coincided with decreased expression/phosphorylation of IGF-1R, AKT and ERK. This cell line also exhibited PPP-induced downregulation of MMP-7 and MMP-9. Similar to the DLD-1 and HCT-116 cell lines, HT-29 also showed substantial cell detachment in response to PPP. Although a net reduction of cells by PPP seems to require a synchronized downregulation of IGF-1R, AKT and ERK1/2, part of the antitumor effect may be explained by other, possibly IGF-1R-unrelated mechanism(s). Such a multitude of inhibitory effects of PPP in colon cancer cells together with its low toxicity in vivo makes it a promising drug candidate in the treatment of this disease.