Therapeutic Exploitation of Checkpoint Defects in Cancer Cells Lacking p53 Function

Cytotoxic agents form the basis of most cancer therapies. These agents primarily affect rapidly proliferating cells, so their use incurs morbidity associated with damage to tissues such as bone marrow and gastrointestinal mucosa. Clinical outcome would be improved if it were possible to develop therapeutics with more specific activity against p53-deficient cancers, which account for over 50% of all cases. p53 deficiency alters the cellular response to DNA damage in that it leaves cells with attenuated DNA damage checkpoint controls and a reduced propensity for apoptotic cell death. Thus, the DNA repair capacity of these cells is reduced but survival is increased. This promotes genomic instability and contributes to the resistance of p53-deficient cells to cytotoxic agents. Disabling the residual G2 checkpoint function of p53-deficient cells may favour cell death following DNA damage. Several potential strategies for G2 checkpoint abrogation show promise for the specific sensitization of cancer cells. Here we detail how the G2 DNA damage checkpoint is influenced by p53 status and how the loss of p53 function in cancer cells can be exploited to enhance the cytotoxicity of anti-cancer agents.     

Anti-Heparanase Aptamers as Potential Diagnostic and Therapeutic Agents for Oral Cancer

Heparanase is an endoglycosidase enzyme present in activated leucocytes, mast cells, placental tissue, neutrophils and macrophages, and is involved in tumour metastasis and tissue invasion. It presents a potential target for cancer therapies and various molecules have been developed in an attempt to inhibit the enzymatic action of heparanase. In an attempt to develop a novel therapeutic with an associated diagnostic assay, we have previously described high affinity aptamers selected against heparanase. In this work, we demonstrated that these anti-heparanase aptamers are capable of inhibiting tissue invasion of tumour cells associated with oral cancer and verified that such inhibition is due to inhibition of the enzyme and not due to other potentially cytotoxic effects of the aptamers. Furthermore, we have identified a short 30 bases aptamer as a potential candidate for further studies, as this showed a higher ability to inhibit tissue invasion than its longer counterpart, as well as a reduced potential for complex formation with other non-specific serum proteins. Finally, the aptamer was found to be stable and therefore suitable for use in human models, as it showed no degradation in the presence of human serum, making it a potential candidate for both diagnostic and therapeutic use.     

Antimicrobial Peptides and their Potential as Oral Therapeutic Agents

Dental caries (tooth decay) and periodontal diseases are the most prevalent bacterial infectious diseases of mankind, together affecting almost the entire population of the world. Both diseases are caused by oral bacteria that exist as components of a polymicrobial biofilm, known as dental plaque, on the tooth surface. The control of specific types of bacteria and/or total numbers of bacteria in dental plaque could lead to prevention or resolution of disease. Antimicrobial peptides isolated from a wide range of natural sources have been known for over 30 years yet little progress had been made in the therapeutic application of these peptides. This is due in part to the characteristics, including susceptibility to proteolysis, of the cationic amphipathic antimicrobial peptides that form the majority of peptides discovered to date. Bovine milk is a readily available source of a range of bioactive peptides. We have isolated and characterized a novel anionic antimicrobial peptide, Kappacin, from bovine milk. Antibacterial activity of the peptide is increased when it is complexed with zinc ions. We have demonstrated that a Kappacin:Zn²⁺ preparation is able to suppress the growth of oral cariogenic bacteria in a biofilm. The Kappacin:Zn²⁺ antibacterial complex may have potential as an additive to oral care products and other delivery vehicles for the control of oral disease.     

Nutlin-3a: A Potential Therapeutic Opportunity for TP53 Wild-Type Ovarian Carcinomas

Epithelial ovarian cancer is a diverse molecular and clinical disease, yet standard treatment is the same for all subtypes. TP53 mutations represent a node of divergence in epithelial ovarian cancer histologic subtypes and may represent a therapeutic opportunity in subtypes expressing wild type, including most low-grade ovarian serous carcinomas, ovarian clear cell carcinomas and ovarian endometrioid carcinomas, which represent approximately 25% of all epithelial ovarian cancer. We therefore sought to investigate Nutlin-3a—a therapeutic which inhibits MDM2, activates wild-type p53, and induces apoptosis—as a therapeutic compound for TP53 wild-type ovarian carcinomas. Fifteen epithelial ovarian cancer cell lines of varying histologic subtypes were treated with Nutlin-3a with determination of IC₅₀ values. Western Blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses quantified MDM2, p53, and p21 expression after Nutlin-3a treatment. DNA from 15 cell lines was then sequenced for TP53 mutations in exons 2-11 including intron-exon boundaries. Responses to Nutlin-3a were dependent upon TP53 mutation status. By qRT-PCR and WB, levels of MDM2 and p21 were upregulated in wild-type TP53 sensitive cell lines, and p21 induction was reduced or absent in mutant cell lines. Annexin V assays demonstrated apoptosis in sensitive cell lines treated with Nutlin-3a. Thus, Nutlin-3a could be a potential therapeutic agent for ovarian carcinomas expressing wild-type TP53 and warrants further investigation.     

Interdependent regulation of p53 and miR-34a in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) has an incidence 4/100 000 people in the western world and is one of the first cancers reported to be associated with deregulated miRNA expression. microRNAs are small non coding RNAs that are important regulators of protein expression through binding to their untranslated 3'-UTR region. The miR-34 family was demonstrated to be induced by the tumor suppressor p53 and to elicit p53-like responses like senescence, cell cycle arrest and apoptosis depending on the cell type. We have shown in a recent paper that miR-34a is severely increased in the TCL1-mouse model of CLL. This finding was reflected in human CLL. Moreover, it is demonstrated that its expression is dependent on the presence of the SNP309 in the intronic promoter of the MDM2 gene. In addition, low miR-34a expression was associated with shorter time to treatment (log-rank P = 0.003) in CLL. When reintroduced into CLL cells, miR-34a was able to induce apoptosis. Interestingly, this was dependent on an intact p53 pathway. Here, we present data showing that knockdown of p53 in HCT-116 cells severely reduces miR-34a induced apoptosis. In conclusion, miR-34a is proposed as a marker for the activity of the p53 pathway in CLL.     

Rb and p53 Liver Functions Are Essential for Xenobiotic Metabolism and Tumor Suppression

The tumor suppressors Retinoblastoma (Rb) and p53 are frequently inactivated in liver diseases, such as hepatocellular carcinomas (HCC) or infections with Hepatitis B or C viruses. Here, we discovered a novel role for Rb and p53 in xenobiotic metabolism, which represent a key function of the liver for metabolizing therapeutic drugs or toxins. We demonstrate that Rb and p53 cooperate to metabolize the xenobiotic 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). DDC is metabolized mainly by cytochrome P450 (Cyp)3a enzymes resulting in inhibition of heme synthesis and accumulation of protoporphyrin, an intermediate of heme pathway. Protoporphyrin accumulation causes bile injury and ductular reaction. We show that loss of Rb and p53 resulted in reduced Cyp3a expression decreased accumulation of protoporphyrin and consequently less ductular reaction in livers of mice fed with DDC for 3 weeks. These findings provide strong evidence that synergistic functions of Rb and p53 are essential for metabolism of DDC. Because Rb and p53 functions are frequently disabled in liver diseases, our results suggest that liver patients might have altered ability to remove toxins or properly metabolize therapeutic drugs. Strikingly the reduced biliary injury towards the oxidative stress inducer DCC was accompanied by enhanced hepatocellular injury and formation of HCCs in Rb and p53 deficient livers. The increase in hepatocellular injury might be related to reduce protoporphyrin accumulation, because protoporphrin is well known for its anti-oxidative activity. Furthermore our results indicate that Rb and p53 not only function as tumor suppressors in response to carcinogenic injury, but also in response to non-carcinogenic injury such as DDC.     

Class I and Class II Histone Deacetylases Are Potential Therapeutic Targets for Treating Pancreatic Cancer

Background

Pancreatic cancer is a highly malignant disease with an extremely poor prognosis. Histone deacetylase inhibitors (HDACIs) have shown promising antitumor activities against preclinical models of pancreatic cancer, either alone or in combination with chemotherapeutic agents. In this study, we sought to identify clinically relevant histone deacetylases (HDACs) to guide the selection of HDAC inhibitors (HDACIs) tailored to the treatment of pancreatic cancer.

Methodology

HDAC expression in seven pancreatic cancer cell lines and normal human pancreatic ductal epithelial cells was determined by Western blotting. Antitumor interactions between class I- and class II-selective HDACIs were determined by MTT assays and standard isobologram/CompuSyn software analyses. The effects of HDACIs on cell death, apoptosis and cell cycle progression, and histone H4, alpha-tubulin, p21, and γH2AX levels were determined by colony formation assays, flow cytometry analysis, and Western blotting, respectively.

Results

The majority of classes I and II HDACs were detected in the pancreatic cancer cell lines, albeit at variable levels. Treatments with MGCD0103 (a class I-selective HDACI) resulted in dose-dependent growth arrest, cell death/apoptosis, and cell cycle arrest in G2/M phase, accompanied by induction of p21 and DNA double-strand breaks (DSBs). In contrast, MC1568 (a class IIa-selective HDACI) or Tubastatin A (a HDAC6-selective inhibitor) showed minimal effects. When combined simultaneously, MC1568 significantly enhanced MGCD0103-induced growth arrest, cell death/apoptosis, and G2/M cell cycle arrest, while Tubastatin A only synergistically enhanced MGCD0103-induced growth arrest. Although MC1568 or Tubastatin A alone had no obvious effects on DNA DSBs and p21 expression, their combination with MGCD0103 resulted in cooperative induction of p21 in the cells.

Conclusion

Our results suggest that classes I and II HDACs are potential therapeutic targets for treating pancreatic cancer. Accordingly, treating pancreatic cancer with pan-HDACIs may be more beneficial than class- or isoform-selective inhibitors.     

TP53 Mutational Status Is a Potential Marker for Risk Stratification in Wilms Tumour with Diffuse Anaplasia

Purpose

The presence of diffuse anaplasia in Wilms tumours (DAWT) is associated with TP53 mutations and poor outcome. As patients receive intensified treatment, we sought to identify whether TP53 mutational status confers additional prognostic information.

Patients and Methods

We studied 40 patients with DAWT with anaplasia in the tissue from which DNA was extracted and analysed for TP53 mutations and 17p loss. The majority of cases were profiled by copy number (n = 32) and gene expression (n = 36) arrays. TP53 mutational status was correlated with patient event-free and overall survival, genomic copy number instability and gene expression profiling.

Results

From the 40 cases, 22 (55%) had TP53 mutations (2 detected only after deep-sequencing), 20 of which also had 17p loss (91%); 18 (45%) cases had no detectable mutation but three had 17p loss. Tumours with TP53 mutations and/or 17p loss (n = 25) had an increased risk of recurrence as a first event (p = 0.03, hazard ratio (HR), 3.89; 95% confidence interval (CI), 1.26–16.0) and death (p = 0.04, HR, 4.95; 95% CI, 1.36–31.7) compared to tumours lacking TP53 abnormalities. DAWT carrying TP53 mutations showed increased copy number alterations compared to those with wild-type, suggesting a more unstable genome (p = 0.03). These tumours showed deregulation of genes associated with cell cycle and DNA repair biological processes.

Conclusion

This study provides evidence that TP53 mutational analysis improves risk stratification in DAWT. This requires validation in an independent cohort before clinical use as a biomarker.     

Tumor Specific Peptides Selected for Targeted Delivery of Therapeutic Agents to Glioma Human Cells

Russian Journal of Bioorganic Chemistry - Brain tumors are among the most intractable types of malignant neoplasms. Despite advances in the treatment of cancer, in particular, the development of...