In vitro quantification of specific microRNA using molecular beacons

MicroRNAs (miRNAs), a class of non-coding RNAs, have become a major focus of molecular biology research because of their diverse genomic origin and ability to regulate an array of cellular processes. Although the biological functions of miRNA are yet to be fully understood, tissue levels of specific miRNAs have been shown to correlate with pathological development of disease. Here, we demonstrate that molecular beacons can readily distinguish mature- and pre-miRNAs, and reliably quantify miRNA expression. We found that molecular beacons with DNA, RNA and combined locked nucleic acid (LNA)–DNA backbones can all detect miRNAs of low (<1 nM) concentrations in vitro, with RNA beacons having the highest detection sensitivity. Furthermore, we found that molecular beacons have the potential to distinguish miRNAs that have slight variations in their nucleotide sequence. These results suggest that the molecular beacon-based approach to assess miRNA expression and distinguish mature and precursor miRNA species is quite robust, and has the promise for assessing miRNA levels in biological samples.     

New photodynamic molecular beacons (PMB) as potential cancer-targeted agents in PDT

Further improvements in Photodynamic therapy (PDT) necessitate that the dye targets more selectively tumour tissues or neovascularization than healthy cells. Different enzymes such as matrix metalloproteinases (MMPs) are overexpressed in tumour areas. Among these MMPs, gelatinases (MMP-2 and MMP-9) and its activator MMP-14 are known to play a key role in tumour angiogenesis and the growth of many cancers such as glioblastoma multiforme (GBM), an aggressive malignant tumour of the brain. These last years, the concept of photodynamic molecular beacons (PMB) became interesting for controlling the photosensitizer’s ability to generate singlet oxygen (¹O₂) close to target biomolecules as MMPs. We report herein novel PMBs triggered by MMP-2 and/or MMP-9 and/or MMP-14, comprising a photosensitizer and a singlet oxygen quencher linked by MMP cleavable peptide linker (H-GRIGFLRTAKGG-OH). First of all, we focused on the synthesis and the photophysical study of different derivatives photosensitizer-peptide. This preliminary work concluded on an influence of the nature and the distance from the peptide, but not of the position of the photosensitizer in these derivatives on the proteolytic enzymatic action. The nature of the quencher used (a blackberry quencher (BBQ-650) or a black hole quencher (BHQ3)) does not influence the enzymatic action. We also studied the influence of an additional PEG spacer. Finally, the synthesis, the singlet oxygen quenching efficiency and the enzymatic activation of these new MMP- cleavable-PMBs were compared.     

Peptide-based molecular beacons for cancer imaging and therapy

Peptide-based molecular beacons are Förster resonance energy transfer-based target-activatable probes. They offer control of fluorescence emission in response to specific cancer targets and thus are useful tools for in vivo cancer imaging. With our increasing knowledge about human genome in health and disease, peptide-based “smart” probes are continually developed for in vivo optical imaging of specific molecular targets, biological pathways and cancer progression and diagnosis. A class of fluorescent photosensitizers further extends the application of peptide beacons to cancer therapeutics. This review highlights the applications of peptide beacons in cancer imaging, the simultaneous treatment and response monitoring and smart therapeutics with a focus on recent improvements in the design of these probes.     

Imaging for distant metastases in women with early-stage breast cancer: a population-based cohort study

Background:

Practice guidelines recommend that imaging to detect metastatic disease not be performed in the majority of patients with early-stage breast cancer who are asymptomatic. We aimed to determine whether practice patterns in Ontario conform with these recommendations.

Methods:

We used provincial registry data to identify a population-based cohort of Ontario women in whom early-stage, operable breast cancer was diagnosed between 2007 and 2012. We then determined whether imaging of the skeleton, thorax, and abdomen or pelvis had been performed within 3 months of tissue diagnosis. We calculated rates of confirmatory imaging of the same body site.

Results:

Of 26 547 patients with early-stage disease, 22 811 (85.9%) had at least one imaging test, and a total of 83 249 imaging tests were performed (mean of 3.7 imaging tests per patient imaged). Among patients with pathologic stage I and II disease, imaging was performed in 79.6% (10 921/13 724) and 92.7% (11 882/12 823) of cases, respectively. Of all imaging tests, 19 784 (23.8%) were classified as confirmatory investigations. Imaging was more likely for patients who were younger, had greater comorbidity, had tumours of higher grade or stage or had undergone preoperative breast ultrasonography, mastectomy or surgery in the community setting.

Interpretation:

Despite recommendations from multiple international guidelines, most Ontario women with early-stage breast cancer underwent imaging to detect distant metastases. Inappropriate imaging in asymptomatic patients with early-stage disease is costly and may lead to harm. The use of population datasets will allow investigators to evaluate whether or not strategies to implement practice guidelines lead to meaningful and sustained change in physician practice.Most women with newly diagnosed breast cancer present with early-stage, potentially curable disease.¹ Among patients whose disease is restricted to the breast and axillary lymph nodes, without signs or symptoms of metastatic disease, the likelihood of having radiologically evident metastases in pathologic stage I and II disease is about 0.2% and 1.2%, respectively.² This low frequency has not changed significantly, even with the increasing use of magnetic resonance imaging (MRI) and positron emission tomography.^2,3 For this reason, most provincial, national and international guidelines do not recommend imaging for all patients with early-stage breast cancer who are asymptomatic.^4–8Despite these evidence-based guidelines, imaging for distant metastases in patients with a new diagnosis of breast cancer remains common.^2,9–12 In response to the Choosing Wisely campaign of the American Board of Internal Medicine Foundation,¹³ the American Society of Clinical Oncology (ASCO) published its inaugural “top 5” list for choosing wisely in oncology.¹⁴ It recommended against routine imaging for staging purposes in women with early breast cancer, because “such imaging adds little benefit to patient care and has the potential to cause harm.”¹⁴ In 2014, Choosing Wisely Canada was launched in an effort to encourage physicians and patients to engage in conversations about unnecessary tests, treatments and procedures, to help ensure that patients receive the highest-quality care.¹⁵The ASCO Choosing Wisely recommendation¹⁴ is similar to the Cancer Care Ontario guideline,⁴ which has been in existence for over a decade. Whereas ASCO in its Choosing Wisely campaign recommends no imaging for patients with stage I or II disease, the Cancer Care Ontario guideline recommends no imaging for patients with stage I disease and a bone scan for those with stage II disease. A recent study at a large Canadian academic cancer centre showed that, despite publication of both a provincial guideline and the ASCO recommendations, most patients with primary operable (early-stage) breast cancer undergo imaging for distant metastases.¹⁰ We hypothesized that despite the provincial guideline, this practice may be more widespread. We undertook this population-based study to determine whether physician practice patterns in Ontario regarding imaging of patients with early-stage breast cancer are in keeping with the published Cancer Care Ontario guideline.     

In vitro selection of molecular beacons

While molecular beacons are primarily known as biosensors for the detection of nucleic acids, it has proven possible to adapt other nucleic acid binding species (aptamers) to function in a manner similar to molecular beacons, yielding fluorescent signals only in the presence of a cognate ligand. Unfortunately, engineering aptamer beacons requires a detailed knowledge of aptamer sequence and structure. In order to develop a general method for the direct selection of aptamer beacons we have first developed a selection method for molecular beacons. A pool of random sequence DNA molecules were immobilized via a capture oligonucleotide on an affinity column, and those variants that could be released from the column by a target oligonucleotide were amplified. After nine rounds of selection and amplification the elution characteristics of the population were greatly improved. A fluorescent reporter in the selected beacons was located adjacent to a DABCYL moiety in the capture oligonucleotide; addition of the target oligonucleotide led to release of the capture oligonucleotide and up to a 17-fold increase in fluorescence. Signaling was specific for the target oligonucleotide, and occurred via a novel mechanism, relative to designed molecular beacons. When the target oligonucleotide is bound it can form a stacked helical junction with an intramolecular hairpin in the selected beacon; formation of the intramolecular hairpin in turn leads to release of the capture oligonucleotide. The ability to select molecular beacons may prove useful for identifying available sites on complex targets, such as mRNAs, while the method for selection can be easily generalized to other, non-nucleic acid target classes.     

Using the singlet oxygen scavenging property of carotenoid in photodynamic molecular beacons to minimize photodamage to non-targeted cells.

We recently introduced the concept of photodynamic molecular beacons (PMB) for selective control of photodynamic therapy (PDT). The PMB consists of a peptide linker that is sequence specific to a cancer-associated protease. A photosensitizer (PS) and a singlet oxygen (1O2) quencher are conjugated to the opposite ends of this linker. Proximity of the PS and quencher can efficiently inhibit 1O2 generation. In the presence of a targeted protease, the substrate sequence is cleaved and the PS and quencher will separate so that the PS can be photo-activated. There are two ways to optimize the PMB selectivity to cancer cells. The first is to increase the protease specificity to targeted cells and the second is to minimize the phototoxicity of intact (uncleaved) PMBs in non-targeted (normal) cells. Carotenoids (CARs) are well known in nature for their role in quenching excited states of PS and in directly scavenging 1O2. The purpose of this study is to evaluate whether the CAR with dual quenching modes (PS excited states deactivation and 1O2 scavenging) can be used to minimize the photodamage of intact PMBs to non-targeted cells. Thus, we synthesized a beacon (PPC) with a caspase-3 cleavable peptide linking a PS and a CAR quencher. It was confirmed that CAR deactivates the PS excited states and also directly scavenges 1O2. Moreover, the in vitro PDT response showed that CAR completely shuts off the photodynamic effect in non-targeted HepG(2) cells, while PS without CAR (control) remains highly potent even at a much lower (30-fold) dose.     

Hybridization kinetics and thermodynamics of molecular beacons

Molecular beacons are increasingly being used in many applications involving nucleic acid detection and quantification. The stem–loop structure of molecular beacons provides a competing reaction for probe–target hybridization that serves to increase probe specificity, which is particularly useful when single-base discrimination is desired. To fully realize the potential of molecular beacons, it is necessary to optimize their structure. Here we report a systematic study of the thermodynamic and kinetic parameters that describe the molecular beacon structure–function relationship. Both probe and stem lengths are shown to have a significant impact on the binding specificity and hybridization kinetic rates of molecular beacons. Specifically, molecular beacons with longer stem lengths have an improved ability to discriminate between targets over a broader range of temperatures. However, this is accompanied by a decrease in the rate of molecular beacon–target hybridization. Molecular beacons with longer probe lengths tend to have lower dissociation constants, increased kinetic rate constants, and decreased specificity. Molecular beacons with very short stems have a lower signal-to-background ratio than molecular beacons with longer stems. These features have significant implications for the design of molecular beacons for various applications.     

Structure-function relationships of shared-stem and conventional molecular beacons

Molecular beacons are oligonucleotide probes capable of forming a stem-loop hairpin structure with a reporter dye at one end and a quencher at the other end. Conventional molecular beacons are designed with a target-binding domain flanked by two complementary short arm sequences that are independent of the target sequence. Here we report the design of shared-stem molecular beacons with one arm participating in both stem formation when the beacon is closed and target hybridization when it is open. We performed a systematic study to compare the behavior of conventional and shared-stem molecular beacons by conducting thermodynamic and kinetic analyses. Shared-stem molecular beacons form more stable duplexes with target molecules than conventional molecular beacons; however, conventional molecular beacons may discriminate between targets with a higher specificity. For both conventional and shared-stem molecular beacons, increasing stem length enhanced the ability to differentiate between wild-type and mutant targets over a wider range of temperatures. Interestingly, probe-target hybridization kinetics were similar for both classes of molecular beacons and were influenced primarily by the length and sequence of the stem. These findings should enable better design of molecular beacons for various applications.     

Graphene oxide-methylene blue nanocomposite in photodynamic therapy of human breast cancer

The interaction of methylene blue (MB) as a photosensitizer with graphene oxide nano-sheets (GO) was examined in aqueous solution using UV-vis spectrophotometric techniques. MB–GO composites were prepared by mixing the solutions of GO nano-sheets and methylene blue due to interacting of the cationic methylene blue photosensitizer via electrostatic and π–π stacking or hydrophobic cooperative interactions. The cell killing potential of nanocomposite was examined on the MDA-MB-231 breast cancer cells in the absence and presence of red LED irradiation. The results demonstrated that the MB-GO nanocomposite has good performance in photodynamic therapy (PDT) during red LED irradiation. The cytotoxicity of nanocomposite caused reducing cell viability up to 20%. These effects would be due to the nano size structure of composite that could lead to effective cellular penetration. Also the significant difference has seen in lower concentrations of MB and MB-GO nanocomposite. The results show more than 40% increases in cell killing potential in lower concentrations of nanocomposite by using 2.5 μg/mL of each compound. The ratio of GO/MB can affect the interaction and higher ratios of graphene oxide (GO/MB > 1) can induce dimerization of MB. In lower concentrations and ratios of (GO/MB < 1) the free MB concentration increases and the electron shuttling effect of GO in photo activity decreases – which could affect the photocatalytic yield in PDT. The cell viability measurements confirm these effects on cancer cell killing potential of nanocomposite. According to microscopic and PDT assay results, the nanocomposite distribution and diffusion in cells enhanced the photochemical reaction yield in photodynamic therapy of MDA-MB-231 breast cancer cell line.     

Multiplex detection of single-nucleotide variations using molecular beacons

We demonstrate that single-nucleotide differences in a DNA sequence can be detected in homogeneous assays using molecular beacons. In this method, the region surrounding the site of a sequence variation is amplified in a polymerase chain reaction and the identity of the variant nucleotide is determined by observing which of four differently colored molecular beacons binds to the amplification product. Each of the molecular beacons is perfectly complementary to one variant of the target sequence and each is labeled with a different fluorophore. To demonstrate the specificity of these assays, we prepared four template DNAs that only differed from one another by the identity of the nucleotide at one position. Four amplification reactions were prepared, each containing all four molecular beacons, but each initiated with only one of the four template DNAs. The results show that in each reaction a fluorogenic response was elicited from the molecular beacon that was perfectly complementary to the amplified DNA, but not from the three molecular beacons whose probe sequence mismatched the target sequence. The color of the fluorescence that appeared in each tube during the course of the amplification indicated which nucleotide was present at the site of variation. These results demonstrate the extraordinary specificity of molecular beacons. Furthermore, the results illustrate how the ability to label molecular beacons with differently colored fluorophores enables simple multiplex assays to be carried out for genetic analysis.     

Pamidronate in the treatment of bone metastases from breast cancer

Bone metastases afflict over 70% of patients with advanced breast cancer, resulting in impaired quality of life and significant clinical problems. Until appearance of the bisphosphonates there was no specific therapeutic treatment available to manage the symptoms of osteolytic bone metastases. Bisphosphonates are stable chemical analogues of pyrophosphate, and inhibit osteoclast-mediated bone resorption, the treatment is effective in reducing skeletal morbidity in breast cancer with fewer skeletal related events, reduced pain and analgesic consumption, and improved quality of life. As a result, bisphosphonates should now be part of the routine management of metastatic bone disease and multiple myeloma. Promising data have resulted in considerable interest in the possible adjuvant use of bisphosphonates. Pamidronate is an easy to use potent inhibitor of osteolysis, given in conjunction with standard anticancer therapies effectively relieves bone pain and improves performance status. Monthly pamidronate infusions for one or two years in addition to standard anticancer therapy reduce by more than one third the yearly frequency of skeletal-related events. The authors report their practice in which 119 breast cancer patients metastatic to bone received 90-120 mg pamidronate infusion/cycle in addition to standard breast cancer therapy every 3-4 weeks.     

Intraoperative cytologic diagnosis of sentinel node metastases in breast cancer

OBJECTIVE: To clarify the usefulness of imprint cytology for intraoperative investigations of sentinel lymph nodes in breast cancer, comparing the results with those of examinations using frozen and permanent sections. STUDY DESIGN: The material consisted of 303 sentinel lymph nodes from 124 cases of clinically node negative breast cancer. Touch imprint cytologic slides and frozen sections were obtained from the same cut surface of the sentinel nodes. Correlations with the final histopathologic results in paraffin sections were evaluated. RESULTS: The sensitivity, specificity and accuracy of imprint cytology were 70.3%, 99.6% and 96.0%, and those of frozen sections were 83.8%, 100%, 98.0%, respectively. The values were improved when the 2 methods were combined (89.2%, 99.6%, 98.3%), though the concordance between imprint cytology and frozen section was 91.9%. CONCLUSION: Both imprint cytology and frozen section are useful for evaluating sentinel lymph node status in breast cancer. However, the 2 techniques should be combined to improve the diagnostic sensitivity.     

Identification of specific reachable molecular targets in human breast cancer using a versatile ex vivo proteomic method

Targeting of tumoral tissues is one of the most promising approaches to improve both the efficacy and safety of anticancer treatments. The identification of valid targets, including proteins specifically and abundantly expressed in cancer lesions, is of utmost importance. Despite state-of-the-art technologies, the discovery of cancer-associated target proteins still faces the limitation, in human tissues, of antigen accessibility to suitable high-affinity ligands such as human mAb bound to bioactive molecules. Terminal perfusion of tumor-bearing mice or ex vivo perfusion of human cancer-bearing organs with a reactive biotin ester solution has successfully led to the identification of novel accessible biomarkers. This methodology is however restricted to perfusable organs, and excludes most of the tissues of interest to targeted therapies, e.g. primary breast cancer and metastases. Herein, we report on the development of a new chemical proteomic method that bypasses the perfusion step and thus offers the potential to identify accessible molecular targets in virtually all types of animal and human tissues. We have validated our new procedure by identifying biomarkers selectively expressed in human breast carcinoma. Overall, this powerful technology may lay the ground not only for custom-made therapies in cancer, but also for the development of therapies that need to be selectively delivered in a specific tissue.     

Dual FRET molecular beacons for mRNA detection in living cells

The ability to visualize in real-time the expression level and localization of specific endogenous RNAs in living cells can offer tremendous opportunities for biological and disease studies. Here we demonstrate such a capability using a pair of molecular beacons, one with a donor and the other with an acceptor fluorophore that hybridize to adjacent regions on the same mRNA target, resulting in fluorescence resonance energy transfer (FRET). Detection of the FRET signal significantly reduced false positives, leading to sensitive imaging of K-ras and survivin mRNAs in live HDF and MIAPaCa-2 cells. FRET detection gave a ratio of 2.25 of K-ras mRNA expression in stimulated and unstimulated HDF, comparable to the ratio of 1.95 using RT–PCR, and in contrast to the single-beacon result of 1.2. We further revealed intriguing details of K-ras and survivin mRNA localization in living cells. The dual FRET molecular beacons approach provides a novel technique for sensitive RNA detection and quantification in living cells.