The brain-penetrant cell-cycle inhibitor p28 sensitizes brain metastases to DNA-damaging agents.

Background: Brain metastases (BMs), the most common tumors of the central nervous system, are life-threatening with a dismal prognosis. The major challenges to developing effective treatments for BMs are the limited abilities of drugs to target tumors and to cross the blood-brain barrier (BBB). We aimed to investigate the efficacy of our therapeutic approach against BMs in mouse models that recapitulate the clinical manifestations of BMs. Methods: BMs mouse models were constructed by injecting human breast, lung cancer, and melanoma intracardially, which allowed the BBB to remain intact. We investigated the ability of the cell-penetrating peptide p28 to cross the BBB in an in vitro 3D model and in the BMs animal models. The therapeutic effects of p28 in combination with DNA-damaging agents (radiation and temozolomide) on BMs were also evaluated. Results: p28 crossed the intact BBB more efficiently than the standard chemotherapeutic agent, temozolomide. Upon crossing the BBB, p28 localized preferentially to tumor lesions and enhanced the efficacy of DNA-damaging agents by activating the p53-p21 axis. In the BMs animal models, radiation in combination with p28 significantly reduced the tumor burden of BMs. Conclusions: The cell-cycle inhibitor p28 can cross the BBB localize to tumor lesions in the brain and enhance the inhibitory effects of DNA-damaging agents on BMs, suggesting the potential therapeutic benefits of this molecule in BMs.

Cross-talk between cancer and Pseudomonas aeruginosa mediates tumor suppression.

Microorganisms living at many sites in the human body compose a complex and dynamic community. Accumulating evidence suggests a significant role for microorganisms in cancer, and therapies that incorporate bacteria have been tried in various types of cancer. We previously demonstrated that cupredoxin azurin secreted by the opportunistic pathogen Pseudomonas aeruginosa, enters human cancer cells and induces apoptotic death1-4. However, the physiological interactions between P. aeruginosa and humans and their role in tumor homeostasis are largely unknown. Here, we show that P. aeruginosa upregulated azurin secretion in response to increasing numbers of and proximity to cancer cells. Conversely, cancer cells upregulated aldolase A secretion in response to increasing proximity to P. aeruginosa, which also correlated with enhanced P. aeruginosa adherence to cancer cells. Additionally, we show that cancer patients had detectable P. aeruginosa and azurin in their tumors and exhibited increased overall survival when they did, and that azurin administration reduced tumor growth in transgenic mice. Our results suggest host-bacterial symbiotic mutualism acting as a diverse adjunct to the host defense system via inter-kingdom communication mediated by the evolutionarily conserved proteins azurin and human aldolase A. This improved understanding of the symbiotic relationship of bacteria with humans indicates the potential contribution to tumor homeostasis.

Tumor-targeting cell-penetrating peptide, p28, for glioblastoma imaging and therapy.

Despite recent advances in cancer research, glioblastoma multiforme (GBM) remains a highly aggressive brain tumor as its treatment options are limited. The current standard treatment includes surgery followed by radiotherapy and adjuvant chemotherapy. However, surgery without image guidance is often challenging to achieve maximal safe resection as it is difficult to precisely discern the lesion to be removed from surrounding brain tissue. In addition, the efficacy of adjuvant chemotherapy is limited by poor penetration of therapeutics through the blood-brain barrier (BBB) into brain tissues, and the lack of tumor targeting. In this regard, we utilized a tumor-targeting cell-penetration peptide, p28, as a therapeutic agent to improve the efficacy of a current chemotherapeutic agent for GBM, and as a carrier for a fluorescence imaging agent for a clear identification of GBM. Here, we show that a near-infrared (NIR) imaging agent, ICG-p28 (a chemical conjugate of an FDA-approved NIR dye, indocyanine green ICG, and tumor-targeting p28 peptide) can preferentially localize tumors in multiple GBM animal models. Moreover, xenograft studies show that p28, as a therapeutic agent, can enhance the cytotoxic activity of temozolomide (TMZ), one of the few effective drugs for brain tumors. Collectively, our findings highlight the important role of the tumor-targeting peptide, which has great potential for intraoperative image-guided surgery and the development of new therapeutic strategies for GBM.

Nontoxic Tumor-Targeting Optical Agents for Intraoperative Breast Tumor Imaging.

Precise identification of the tumor margins during breast-conserving surgery (BCS) remains a challenge given the lack of visual discrepancy between malignant and surrounding normal tissues. Therefore, we developed a fluorescent imaging agent, ICG-p28, for intraoperative imaging guidance to better aid surgeons in achieving negative margins in BCS. Here, we determined the pharmacokinetics (PK), biodistribution, and preclinical toxicity of ICG-p28. The PK and biodistribution of ICG-p28 indicated rapid tissue uptake and localization at tumor lesions. There were no dose-related effect and no significant toxicity in any of the breast cancer and normal cell lines tested. Furthermore, ICG-p28 was evaluated in clinically relevant settings with transgenic mice that spontaneously developed invasive mammary tumors. Intraoperative imaging with ICG-p28 showed a significant reduction in the tumor recurrence rate. This simple, nontoxic, and cost-effective method can offer a new approach that enables surgeons to intraoperatively identify tumor margins and potentially improves overall outcomes by reducing recurrence rates.

Image-guided surgery with a new tumour-targeting probe improves the identification of positive margins.

BACKGROUND: Given the lack of visual discrepancy between malignant and surrounding normal tissue, current breast conserving surgery (BCS) is associated with a high re-excision rate. Due to the increasing cases of BCS, a novel method of complete tumour removal at the initial surgical resection is critically needed in the operating room to help optimize the surgical procedure and to confirm tumour-free edges. METHODS: We developed a unique near-infrared (NIR) fluorescence imaging probe, ICG-p28, composed of the clinically nontoxic tumour-targeting peptide p28 and the FDA-approved NIR dye indocyanine green (ICG). ICG-p28 was characterized in vitro and evaluated in multiple breast cancer animal models with appropriate control probes. Our experimental approach with multiple-validations and -blinded procedures was designed to determine whether ICG-p28 can accurately identify tumour margins in mimicked intraoperative settings. FINDINGS: The in vivo kinetics were analysed to optimize settings for potential clinical use. Xenograft tumours stably expressing iRFP as a tumour marker showed significant colocalization with ICG-p28, but not ICG alone. Image-guided surgery with ICG-p28 showed an over 6.6 × 103-fold reduction in residual normalized tumour DNA at the margin site relative to control approaches (i.e., surgery with ICG or palpation/visible inspection alone), resulting in an improved tumour recurrence rate (92% specificity) in multiple breast cancer animal models independent of the receptor expression status. ICG-p28 allowed accurate identification of tumour cells in the margin to increase the complete resection rate. INTERPRETATION: Our simple and cost-effective approach has translational potential and offers a new surgical procedure that enables surgeons to intraoperatively identify tumour margins in a real-time, 3D fashion and that notably improves overall outcomes by reducing re-excision rates. FUNDING: This work was supported by NIH/ National Institute of Biomedical Imaging and Bioengineering, R01EB023924.

A Method of Tumor In Vivo Imaging with a New Peptide-Based Fluorescent Probe.

Precise surgical resection directly influences the prognosis and survival of patients with solid tumors. However, it is often difficult to distinguish tumor from normal tissue during resection without any intraoperative imaging guidance. Image-guided surgery particularly when coupled with a near-infrared (NIR) fluorescent agent may improve positive-margin rate thereby improving the overall prognosis. We have developed a unique tumor-targeting fluorescence imaging agent that can aid in the accurate localization of human cancer cells in preclinical settings. The NIR imaging agent, ICG-p28, a water-soluble, nontoxic, and pan-tumor targeting probe consisting of a cell-penetrating peptide (p28) conjugated to indocyanine green (ICG), can accurately localize tumors in vivo. Development of the noninvasive, targeted imaging agent can potentially improve in the resections of tumors by enabling the localization of lesions that are currently difficult or impossible to detect by visual observation or palpation. Here, we describe the methods of preclinical animal imaging models by using NIR fluorescence imager coupled with a new tumor-targeting agent.

p28-Mediated Activation of p53 in G2-M Phase of the Cell Cycle Enhances the Efficacy of DNA Damaging and Antimitotic Chemotherapy.

p28 is an anionic cell-penetrating peptide of 28 amino acids that activates wild-type and mutated p53, leading subsequently to selective inhibition of CDK2 and cyclin A expression and G2-M cell-cycle arrest. In this study, we investigated the cytotoxic effects of p28 treatment alone and in combination with DNA-damaging and antimitotic agents on human cancer cells. p28 enhanced the cytotoxic activity of lower concentrations (IC20-50) of DNA-damaging drugs (doxorubicin, dacarbazine, temozolamide) or antimitotic drugs (paclitaxel and docetaxel) in a variety of cancer cells expressing wild-type or mutated p53. Mechanistic investigations revealed that p28 induced a post-translational increase in the expression of wild-type or mutant p53 and p21, resulting in cell-cycle inhibition at the G2-M phase. The enhanced activity of these anticancer agents in combination with p28 was facilitated through the p53/p21/CDK2 pathway. Taken together, these results highlight a new approach to maximize the efficacy of chemotherapeutic agents while reducing dose-related toxicity. Cancer Res; 76(8); 2354-65. ©2016 AACR.

p28, an anionic cell-penetrating peptide, increases the activity of wild type and mutated p53 without altering its conformation.

p28, a cell penetrating peptide, binds to the DNA binding domain (DBD) of p53, inducing a post-translational increase in intracellular levels of wild type and mutant p53 activating pathways that inhibit cancer cell proliferation at G2/M. Cancer cells respond to p28 with an increase in p53 activity, except when mutations either alter DNA contact or completely unfold the DBD. The increase in p53 activity is accompanied by a significant reduction in the level of the E3 ligase COP1, with no alteration in p53 conformation. This suggests p28 can activate p53 over a wide range of conformational mutations by inhibiting the binding of COP1 to p53.

MADD knock-down enhances doxorubicin and TRAIL induced apoptosis in breast cancer cells.

The Map kinase Activating Death Domain containing protein (MADD) isoform of the IG20 gene is over-expressed in different types of cancer tissues and cell lines and it functions as a negative regulator of apoptosis. Therefore, we speculated that MADD might be over-expressed in human breast cancer tissues and that MADD knock-down might synergize with chemotherapeutic or TRAIL-induced apoptosis of breast cancer cells. Analyses of breast tissue microarrays revealed over-expression of MADD in ductal and invasive carcinomas relative to benign tissues. MADD knockdown resulted in enhanced spontaneous apoptosis in human breast cancer cell lines. Moreover, MADD knockdown followed by treatment with TRAIL or doxorubicin resulted in increased cell death compared to either treatment alone. Enhanced cell death was found to be secondary to increased caspase-8 activation. These data indicate that strategies to decrease MADD expression or function in breast cancer may be utilized to increase tumor cell sensitivity to TRAIL and doxorubicin induced apoptosis.

Prosthodontic rehabilitation of velopharyngeal disorders-a case series.

Acquired palatal defects results from cancer resection, whereas cleft palate is the main cause of congenital defects. Both these condition leads to impaired speech intelligibility, seepage of nasal secretions into the oral cavity and vice versa while difficulty in swallowing and hyper nasality is also eminent. In this case report three different technique for treating velopharyngeal disorders have been described.

Implant supported prosthesis after ridge augmentation procedure by distraction osteogenesis for atrophic mandible.

Rehabilitation of deficient alveolar ridges has long been a challenge. The distraction osteogenesis procedure before placement of dental implants has solved the puzzle and its advantages over block grafting includes simultaneous expansion of soft tissue, high degree of dimensional stability, abbreviated overall treatment time, and no graft required. In this case report distraction osteogenesis of deficient anterior mandibular ridge was performed and then an implant supported fixed prosthesis was fabricated.

Magnet retained intraoral-extra oral combination prosthesis: a case report.

Facial prosthesis is generally considered over surgical reconstruction to restore function and appearance in patients with facial defects that resulted from cancer resection. Retention of the prosthesis is challenging due to its size and weight. Retention can be achieved by using medical grade adhesives, resilient attachments, clips and osseointegrated implants. It can also be connected to obturator by magnets. This clinical report highlights the rehabilitation of a lateral midfacial defect with a two piece prosthesis that included an extra oral facial prosthesis and an intraoral obturator with the use of magnets.

Indirect resin-bonded fiber-reinforced composite anterior bridge: A case report.

The loss of anterior teeth can be painful for patients, both psychologically and socially. In recent years, fiber-reinforced composite resins (FRCs) have gained increasing acceptance in mainstream prosthodontics as viable alternatives to alloy-based restorations. A combination of good esthetics and fracture toughness validates their use in a diverse range of clinical applications. In this study, a bridge was constructed with hybrid composite resin placed around a resin-impregnated, plasma-etched, polyalkene fiber bonded to the abutment tooth enamel using the acid-etch technique. This method of tooth replacement is viewed as a fast, simple, cost-effective means of replacing anterior teeth. The restoration remained intact, with no discoloration or deterioration at the recall visits. While definitive long-term data about their clinical performance is scarce, FRC prostheses appear to have a worthwhile role in the conservative treatment of short, edentulous spaces.

Full-mouth rehabilitation of a patient with severe attrition using the Hobo twin-stage procedure.

Excessive occlusal wear can result in pulpal injury, occlusal disharmony, impaired function and aesthetic deformity. Loss of anterior guidance can result from severe wear of anterior teeth, which protects the posterior teeth during excursive movement. The collapse of posterior teeth also results in the loss of normal occlusal plane and the reduction of the vertical dimension. This clinical report describes the use of the Hobo twin-stage procedure for rehabilitation of a patient with severe tooth wear, resulting in reduced vertical dimension of occlusion.

A simple, time-saving chairside device for radiographic diagnosis of vertical bone height and soft tissue thickness for implant placement--clinical report.

A diagnostic radiograph of the posterior regions of the jaws is mandatory if implant placement is considered at these sites. The goal is to avoid damage to important anatomical structures such as the inferior alveolar canal and maxillary sinus. Using radiographs to evaluate the remaining bone height available for implant insertion is a common procedure. Many articles have evaluated the success rates of fixed prostheses supported by osseointegrated implants, indicating high rates of success for prostheses 5-10 years in function. However, if an orthopantomogram machine is not readily available, periapical radiographs can be used for vertical bone height assessment. The familiar procedure of incorporating a metal ball of known diameter into an acrylic template is the method of choice. This is a relatively tedious and expensive procedure involving multiple steps, including impression taking, creation of a study model, and manufacture of a metal ball-bearing acrylic template. A further disadvantage for the patient is that a diagnostic radiograph can be taken only at the next appointment. This paper describes a quick and simple method to obtain a radiographic evaluation of remaining alveolar bone height and soft tissue thickness prior to implant placement with help of a metal ball device to avoid template fabrication and time consumption. Soft tissue thickness is measured nonsurgically.

A cell penetrating peptide derived from azurin inhibits angiogenesis and tumor growth by inhibiting phosphorylation of VEGFR-2, FAK and Akt.

Amino acids 50-77 (p28) of azurin, a 128 aa cupredoxin isolated from Pseudomonas aeruginosa, is essentially responsible for azurin's preferential penetration of cancer cells. We now report that p28 also preferentially penetrates human umbilical vein endothelial cells (HUVEC), co-localized with caveolin-1 and VEGFR-2, and inhibits VEGF- and bFGF-induced migration, capillary tube formation and neoangiogenesis in multiple xenograft models. The antiangiogenic effect of p28 in HUVEC is associated with a dose-related non-competitive inhibition of VEGFR-2 kinase activity. However, unlike other antiangiogenic agents that inhibit the VEGFR-2 kinase, p28 decreased the downstream phosphorylation of FAK and Akt that normally precedes cellular repositioning of the cytoskeletal (F-actin), focal adhesion (FAK and paxillin), and cell to cell junction protein PECAM-1, inhibiting HUVEC motility and migration. The decrease in pFAK and pAkt levels suggests that p28 induces a pFAK-mediated loss of HUVEC motility and migration and a parallel Akt-associated reduction in cell matrix attachment and survival. This novel, direct antiangiogenic effect of p28 on endothelial cells may enhance the cell cycle inhibitory and apoptotic properties of this prototype peptide on tumor cell proliferation as it enters a Phase II clinical trial.

Non-rigid connector: The wand to allay the stresses on abutment.

The use of rigid connectors in 5-unit fixed dental prosthesis with a pier abutment can result in failure of weaker retainer in the long run as the pier abutment acts as a fulcrum. Non-rigid connector placed on the distal aspect of pier seems to reduce potentially excess stress concentration on the pier abutment.

A Study on Effect of Surface Treatments on the Shear Bond Strength between Composite Resin and Acrylic Resin Denture Teeth.

Visible light-cured composite resins have become popular in prosthetic dentistry for the replacement of fractured/debonded denture teeth, making composite denture teeth on partial denture metal frameworks, esthetic modification of denture teeth to harmonize with the characteristics of adjacent natural teeth, remodelling of worn occlusal surfaces of posterior denture teeth etc. However, the researches published on the bond strength between VLC composite resins and acrylic resin denture teeth is very limited. The purpose of this study is to investigate the effect of five different methods of surface treatments on acrylic resin teeth on the shear bond strength between light activated composite resin and acrylic resin denture teeth. Ninety cylindrical sticks of acrylic resin with denture teeth mounted atop were prepared. Various treatments were done upon the acrylic resin teeth surfaces. The samples were divided into six groups, containing 15 samples each. Over all the treated and untreated surfaces of all groups, light-cured composite resin was applied. The shear strengths were measured in a Universal Testing Machine using a knife-edge shear test. Data were analyzed using one way analysis of variance (ANOVA) and mean values were compared by the F test. Application of bonding agent with prior treatment of methyl methacrylate on the acrylic resin denture teeth resulted in maximum bond strength with composite resin.

Preclinical pharmacokinetics, metabolism, and toxicity of azurin-p28 (NSC745104) a peptide inhibitor of p53 ubiquitination.

PURPOSE: Characterize the preclinical pharmacokinetics, metabolic profile, multi-species toxicology, and antitumor efficacy of azurin-p28 (NSC 745104), an amphipathic, 28 amino acid fragment (aa 50-77) of the copper containing redox protein azurin that preferentially enters cancer cells and is currently under development for treatment of p53-positive solid tumors. METHODS: An LC/MS/MS assay was developed, validated, and applied to liver microsomes, serum, and tumor cells to assess cellular uptake and metabolic stability. Pharmacokinetics was established after administration of a single intravenous dose of p28 in preclinical species undergoing chronic toxicity testing. Antitumor efficacy was assessed on human tumor xenografts. A human therapeutic dose was predicted based on efficacy and pharmacokinetic parameters. RESULTS: p28 is stable, showed tumor penetration consistent with selective entry into tumor cells and significantly inhibited p53-positive tumor growth. Renal clearance, volume of distribution, and metabolic profile of p28 was relatively similar among species. p28 was non-immunogenic and non-toxic in mice and non-human primates (NHP). The no observed adverse effect level (NOAEL) was 120 mg/kg iv in female mice. A NOAEL was not established for male mice due to decreased heart and thymus weights that was reversible and did not result in limiting toxicity. In contrast, the NOAEL for p28 in NHP was defined as the highest dose (120 mg/kg/dose; 1,440 mg/m(2)/dose) studied. The maximum-tolerated dose (MTD) for subchronic administration of p28 to mice is >240 mg/kg/dose (720 mg/m(2)/dose), while the MTD for subchronic administration of p28 to Cynomolgous sp. is >120 mg/kg (1,440 mg/m(2)/dose). The efficacious (murine) dose of p28 was 10 mg/kg ip per day. CONCLUSIONS: p28 does not exhibit preclinical immunogenicity or toxicity, has a similar metabolic profile among species, and is therapeutic in xenograft models.

A 28-amino-acid peptide fragment of the cupredoxin azurin prevents carcinogen-induced mouse mammary lesions.

Azurin, a member of the cupredoxin family of redox proteins, preferentially penetrates human cancer cells and exerts cytostatic and apoptotic effects. Azurin and amino acids 50-77 (p28) of azurin also produce a dose-dependent reduction in the proliferation of human mammary cancer by increasing the level of the tumor suppressor protein p53 in the cancer cell nucleus. We show that the development of 7,12-dimethylbenz[a]anthracene-induced hormone-dependent premalignant mammary ductal lesions and hormone-independent mammary alveolar lesions in mouse mammary gland organ culture is also significantly reduced by azurin and p28. The dose-dependent reduction in carcinogen-induced mammary cell proliferation by p28 was associated with an increase in the expression of p53. p28 also enhanced the inhibitory effect of a low dose of the antiestrogen tamoxifen on the development of hormone-dependent mammary ductal lesions, but did not enhance the inhibitory activity of fenretinide (N-4-hydroxyphenyl retinamide) on hormone-independent mammary alveolar lesions. These observations suggest that cupredoxins and fragments derived from them can exert a chemopreventive effect on carcinogen-induced mammary gland transformation, irrespective of hormonal environment, and enhance the inhibitory effects of tamoxifen in this model of preneoplastic mammary development.