An 19F NMR fragment-based approach for the discovery and development of BRCA2-RAD51 inhibitors to pursuit synthetic lethality in combination with PARP inhibition in pancreatic cancer.

The BRCA2-RAD51 interaction remains an intriguing target for cancer drug discovery due to its vital role in DNA damage repair mechanisms, which cancer cells become particularly reliant on. Moreover, RAD51 has many synthetically lethal partners, including PARP1-2, which can be exploited to induce synthetic lethality in cancer. In this study, we established a 19F-NMR-fragment based approach to identify RAD51 binders, leading to two initial hits. A subsequent SAR program identified 46 as a low micromolar inhibitor of the BRCA2-RAD51 interaction. 46 was tested in different pancreatic cancer cell lines, to evaluate its ability to inhibit the homologous recombination DNA repair pathway, mediated by BRCA2-RAD51 and trigger synthetic lethality in combination with the PARP inhibitor talazoparib, through the induction of apoptosis. Moreover, we further analyzed the 46/talazoparib combination in 3D pancreatic cancer models. Overall, 46 showed its potential as a tool to evaluate the RAD51/PARP1-2 synthetic lethality mechanism, along with providing a prospect for further inhibitors development.

Preomic profile of BxPC-3 cells after treatment with BRC4.

BRCA2 and RAD51 are two proteins that play a central role in homologous recombination (HR) and DNA double strand break (DSB) repair. BRCA2 assists RAD51 fibrillation and defibrillation through binding with its eight BRC repeats, with BRC4 being one of the most efficient and best characterized. RAD51 inactivation by small molecules has been proposed as a strategy to impair BRCA2/RAD51 binding and, ultimately, the HR pathway, with the aim of making cancer cells more sensitive to PARP inhibitors (PARPi). This strategy, which mimics a synthetic lethality (SL) approach, has been successfully performed in vitro by using the myristoylated derivative of BRC4 (myr-BRC4), designed for a more efficient cell entry. The present study applies a method to obtain a proteomic fingerprint after cellular treatment with the myr-BRC4 peptide using a mass spectroscopy (MS) proteomic approach. (Data are available via ProteomeXchange with identifier PXD042696.) We performed a comparative proteomic profiling of the myr-BRC4 treated vs. untreated BxPC-3 pancreatic cancer cells and evaluated the differential expression of proteins. Among the identified proteins, we focused our attention on proteins shared by both the RAD51 and the BRCA2 interactomes, and on those whose reduction showed high statistical significance. Three downregulated proteins were identified (FANCI, FANCD2, and RPA3), and protein downregulation was confirmed through immunoblotting analysis, validating the MS approach. Our results suggest that, being a direct consequence of myr-BRC4 treatment, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring HR impairment. SIGNIFICANCE: RAD51's inhibition has gained increasing attention because of its possible implications in personalized medicine through the SL approach. Chemical disruption of protein-protein interactions (PPIs) between RAD51 and BRCA2, or some of its partner proteins, could potentiate PARPi DNA damage-induced cell death. This could have application for difficult to treat cancers, such as BRCA-competent and olaparib (PARPi) resistant pancreatic adenocarcinoma. Despite RAD51 being a widely studied target, researchers still lack detailed mechanistic information. This has stifled progress in the field with only a few RAD51 inhibitors having been identified, none of which have gained regulatory approval. Nevertheless, the peptide BRC4 is one of the most specific and best characterized RAD51 binder and inhibitor reported to date. Our study is the first to report the proteomic fingerprint consequent to cellular treatment of myr-BRC4, to offer a reference for the discovery of specific protein/pathway alterations within DNA damage repair. Our results suggest that, being a direct consequence of myr-BRC4 treatment, and ultimately ofBRCA2/RAD51 disruption, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring DNA damage repair impairment and therefore be used to potentiate the development of new effective therapeutic strategies.

Lactate Can Modulate the Antineoplastic Effects of Doxorubicin and Relieve the Drug's Oxidative Damage on Cardiomyocytes.

BACKGROUND: Doxorubicin (DOXO) is currently administered as the first-choice therapy for a variety of malignancies. Cancer cells exhibit enhanced glycolysis and lactate production. This metabolite affects gene expression and can play a role in chemoresistance. AIM OF THIS STUDY: We investigated whether the enhanced lactate levels that characterize neoplastic tissues can modify the response of cancer cells to DOXO. METHODS: After exposing cancer cells to increased lactate levels, we examined whether this metabolite could interfere with the principal mechanisms responsible for the DOXO antineoplastic effect. RESULTS: Increased lactate levels did not affect DOXO-induced topoisomerase poisoning but offered protection against the oxidative damage caused by the drug. This protection was related to changes in gene expression caused by the combined action of DOXO and lactate. Oxidative damage significantly contributed to the heavy cardiotoxicity following DOXO treatment. In cultured cardiomyocytes, we confirmed that DOXO-induced DNA damage and oxidative stress can be significantly mitigated by exposing the cells to increased lactate levels. CONCLUSIONS: In addition to contributing to elucidating the effects of the combined action of DOXO and lactate, our results suggest a possible method to reduce the heavy drug cardiotoxicity, a major side effect leading to therapy discontinuation.

Balance Rehabilitation through Robot-Assisted Gait Training in Post-Stroke Patients: A Systematic Review and Meta-Analysis.

BACKGROUND: Balance impairment is a common disability in post-stroke survivors, leading to reduced mobility and increased fall risk. Robotic gait training (RAGT) is largely used, along with traditional training. There is, however, no strong evidence about RAGT superiority, especially on balance. This study aims to determine RAGT efficacy on balance of post-stroke survivors. METHODS: PubMed, Cochrane Library, and PeDRO databases were investigated. Randomized clinical trials evaluating RAGT efficacy on post-stroke survivor balance with Berg Balance Scale (BBS) or Timed Up and Go test (TUG) were searched. Meta-regression analyses were performed, considering weekly sessions, single-session duration, and robotic device used. RESULTS: A total of 18 trials have been included. BBS pre-post treatment mean difference is higher in RAGT-treated patients, with a pMD of 2.17 (95% CI 0.79; 3.55). TUG pre-post mean difference is in favor of RAGT, but not statistically, with a pMD of -0.62 (95%CI - 3.66; 2.43). Meta-regression analyses showed no relevant association, except for TUG and treatment duration (ß = -1.019, 95% CI - 1.827; -0.210, p-value = 0.0135). CONCLUSIONS: RAGT efficacy is equal to traditional therapy, while the combination of the two seems to lead to better outcomes than each individually performed. Robot-assisted balance training should be the focus of experimentation in the following years, given the great results in the first available trials. Given the massive heterogeneity of included patients, trials with more strict inclusion criteria (especially time from stroke) must be performed to finally define if and when RAGT is superior to traditional therapy.

The Mechanistic Understanding of RAD51 Defibrillation: A Critical Step in BRCA2-Mediated DNA Repair by Homologous Recombination.

The cytotoxic action of anticancer drugs can be potentiated by inhibiting DNA repair mechanisms. RAD51 is a crucial protein for genomic stability due to its critical role in the homologous recombination (HR) pathway. BRCA2 assists RAD51 fibrillation and defibrillation in the cytoplasm and nucleus and assists its nuclear transport. BRC4 is a peptide derived from the fourth BRC repeat of BRCA2, and it lacks the nuclear localization sequence. Here, we used BRC4 to (i) reverse RAD51 fibrillation; (ii) avoid the nuclear transport of RAD51; and (iii) inhibit HR and enhance the efficacy of chemotherapeutic treatments. Specifically, using static and dynamic light scattering, transmission electron microscopy, and microscale thermophoresis, we show that BRC4 eroded RAD51 fibrils from their termini through a "domino" mechanism and yielded monomeric RAD51 with a cumulative nanomolar affinity. Using cellular assays (BxPC-3, pancreatic cancer), we show that a myristoylated BRC4 (designed for a more efficient cell entry) abolished the formation of nuclear RAD51 foci. The present study provides a molecular description of RAD51 defibrillation, an essential step in BRCA2-mediated homologous recombination and DNA repair.

Lactate is a potential promoter of tamoxifen resistance in MCF7 cells.

BACKGROUND: Tamoxifen is a widely used estrogen receptor inhibitor, whose clinical success is limited by the development of acquired resistance. This compound was also found to inhibit mitochondrial function, causing increased glycolysis and lactate production. Lactate has been widely recognized as a signaling molecule, showing the potential of modifying gene expression. These metabolic effects of tamoxifen can by hypothesized to contribute in driving drug resistance. METHODS: To test this hypothesis, we used MCF7 cells together with a tamoxifen resistant cell line (MCF7-TAM). Experiments were aimed at verifying whether enhanced lactate exposure can affect the phenotype of MCF7 cells, conferring them features mirroring those observed in the tamoxifen resistant culture. RESULTS: The obtained results suggested that enhanced lactate in MCF7 cells medium can increase the expression of tafazzin (TAZ) and telomerase complex (TERC, TERT) genes, reducing the cells' attitude to undergo senescence. In long term lactate-exposed cells, signs of EGFR activation, a pathway related to acquired tamoxifen resistance, was also observed. CONCLUSIONS: The obtained results suggested lactate as a potential promoter of tamoxifen resistance. The off-target effects of this compound could play a role in hindering its therapeutic efficacy. GENERAL SIGNIFICANCE: The features of acquired tamoxifen resistance have been widely characterized at the molecular level; in spite of their heterogeneity, poorly responsive cells were often found to display upregulated glycolysis. Our results suggest that this metabolic asset is not simply a result of neoplastic progression, but can play an active part in driving this process.

Allosteric transitions of rabbit skeletal muscle lactate dehydrogenase induced by pH-dependent dissociation of the tetrameric enzyme.

Among the functions exerted by eukaryotic lactate dehydrogenases, it is of importance the generation of lactate in muscles subjected to fatigue or to limited oxygen availability, with both these conditions triggering a decrease of cellular pH. However, the mutual dependence between lactate dehydrogenase (LDH) catalytic action and lactic acidosis is far from being fully understood. Here we show that the tetrameric LDH from rabbit skeletal muscle undergoes allosteric transitions as a function of pH, i.e. the enzyme obeys Michaelis-Menten kinetics at neutral or slightly alkaline pH values, and features sigmoidal kinetics at pH 6.5 or lower. Remarkably, we also report that a significant dissociation of tetrameric rabbit LDH occurs under acidic conditions, with pyruvate/NAD+ or citrate counteracting this effect. Moreover, citrate strongly activates rabbit LDH, inducing the enzyme to feature Michaelis-Menten kinetics. Further, using primary rabbit skeletal muscle cells we tested the generation of lactate as a function of pH, and we detected a parallel decrease of cytosolic pH and secretion of lactate. Overall, our observations indicate that lactic acidosis is antagonized by LDH dissociation, the occurrence of which is regulated by citrate and by allosteric transitions of the enzyme induced by pyruvate.

Human lactate dehydrogenase A undergoes allosteric transitions under pH conditions inducing the dissociation of the tetrameric enzyme.

The aerobic energetic metabolism of eukaryotic cells relies on the glycolytic generation of pyruvate, which is subsequently channelled to the oxidative phosphorylation taking place in mitochondria. However, under conditions limiting oxidative phosphorylation, pyruvate is coupled to alternative energetic pathways, e.g. its reduction to lactate catalyzed by lactate dehydrogenases (LDHs). This biochemical process is known to induce a significant decrease in cytosolic pH, and is accordingly denoted lactic acidosis. Nevertheless, the mutual dependence of LDHs action and lactic acidosis is far from being fully understood. Using human LDH-A, here we show that when exposed to acidic pH this enzyme is subjected to homotropic allosteric transitions triggered by pyruvate. Conversely, human LDH-A features Michaelis-Menten kinetics at pH values equal to 7.0 or higher. Further, citrate, isocitrate, and malate were observed to activate human LDH-A, both at pH 5.0 and 6.5, with citrate and isocitrate being responsible for major effects. Dynamic light scattering (DLS) experiments revealed that the occurrence of allosteric kinetics in human LDH-A is mirrored by a consistent dissociation of the enzyme tetramer, suggesting that pyruvate promotes tetramer association under acidic conditions. Finally, using the human liver cancer cell line HepG2 we isolated cells featuring cytosolic pH equal to 7.3 or 6.5, and we observed a concomitant decrease in cytosolic pH and lactate secretion. Overall, our observations indicate the occurrence of a negative feedback between lactic acidosis and human LDH-A activity, and a complex regulation of this feedback by pyruvate and by some intermediates of the Krebs cycle.

Effects of robot-assisted gait training on postural instability in Parkinson's disease: a systematic review.

INTRODUCTION: Postural instability is a cardinal feature of Parkinson's disease, together with rest tremor, rigidity and bradykinesia. It is a highly disabling symptom that becomes increasingly common with disease progression and represents a major source of reduced quality of life in patients with Parkinson's disease. Rehabilitation aims to enable patients with Parkinson's disease to maintain their maximum level of mobility, activity and independence. To date, a wide range of rehabilitation approaches has been employed to treat postural instability in Parkinson's disease, including robotic training. Our main aim was to conduct a systematic review of current literature about the effects of robot-assisted gait training on postural instability in patients with Parkinson's disease. EVIDENCE ACQUISITION: A systematic search using the following MeSH terms "Parkinson disease," "postural balance," "robotics," "rehabilitation" AND string "robotics [mh]" OR "robot-assisted" OR "electromechanical" AND "rehabilitation [mh]" OR "training" AND "postural balance [mh]" was conducted on PubMed, Cochrane Library and Pedro electronic databases. Full text articles in English published up to December 2020 were included. Data about patient characteristics, robotic devices, treatment procedures and outcome measures were considered. Every included article got checked for quality. Level of evidence was defined for all studies. EVIDENCE SYNTHESIS: Three authors independently extracted and verified data. In total, 18 articles (2 systematic reviews, 9 randomized controlled trials, 4 uncontrolled studies and 3 case series/case reports) were included. Both end-effector and exoskeleton devices were investigated as to robot-assisted gait training modalities. No clear relationship between treatment parameters and clinical conditions was observed. We found a high level of evidence about the effects of robot-assisted gait training on balance and freezing of gait in patients with Parkinson's disease. CONCLUSIONS: This systematic review provides to the reader a complete overview of current literature and levels of evidence about the effects of robot-assisted gait training on postural instability issues (static and dynamic balance, freezing of gait, falls, confidence in activities of daily living and gait parameters related to balance skills) in patients with Parkinson's disease.

Lactate Upregulates the Expression of DNA Repair Genes, Causing Intrinsic Resistance of Cancer Cells to Cisplatin.

Intrinsic or acquired drug resistance is one of the major problems compromising the success of antineoplastic treatments. Several evidences correlated some therapeutic failures with changes in cell metabolic asset and in line with these findings, hindering the glycolytic metabolism of cancer cells via lactate dehydrogenase (LDH) inhibition was found to overcome the resistance to chemotherapeutic agents. Lactate, the product of LDH reaction, was shown to be involved in epigenetic regulation of gene expression. The experiments described in this paper were aimed at highlighting a possible direct effect of lactate in modifying the response of cancer cells to a chemotherapeutic treatment. To discriminate between the effects potentially caused by glycolytic metabolism from those directly referable to lactate, we selected cancer cell lines able to grow in glucose deprived conditions and evaluated the impact of lactate on the cellular response to cisplatin-induced DNA damage. In lactate-exposed cells we observed a reduced efficacy of cisplatin, which was associated with reduced signatures of DNA damage, enhanced DNA recombination competence and increased expression of a panel of genes involved in DNA repair. The identified genes take part in mismatch and nucleotide excision repair pathways, which were found to contribute in restoring the cisplatin-induced DNA damage. The obtained results suggest that this metabolite could play a role in reducing the efficacy of antineoplastic treatments.

Lactate dehydrogenase inhibition affects homologous recombination repair independently of cell metabolic asset; implications for anticancer treatment.

BACKGROUND: Cancer cells show highly increased glucose utilization which, among other cancer-essential functions, was found to facilitate DNA repair. Lactate dehydrogenase (LDH) activity is pivotal for supporting the high glycolytic flux of cancer cells; to our knowledge, a direct contribution of this enzyme in the control of DNA integrity was never investigated. In this paper, we looked into a possible LDH-mediated regulation of homologous recombination (HR) repair. METHODS: We identified two cancer cell lines with different assets in energy metabolism: either based on glycolytic ATP or on oxidative reactions. In cells with inhibited LDH, we assessed HR function by applying four different procedures. RESULTS: Our findings revealed an LDH-mediated control of HR, which was observed independently of cell metabolic asset. Since HR inhibition is known to make cancer cells responsive to PARP inhibitors, in both the cellular models we finally explored the effects of a combined inhibition of LDH and PARP. CONCLUSIONS: The obtained results suggest for LDH a central role in cancer cell biology, not merely linked to the control of energy metabolism. The involvement of LDH in the DNA damage response could suggest new drug combinations to obtain improved antineoplastic effects. GENERAL SIGNIFICANCE: Several evidences have correlated the metabolic features of cancer cells with drug resistance and LDH inhibition has been repeatedly shown to increase the antineoplastic power of chemotherapeutics. By shedding light on the processes linking cell metabolism to the control of DNA integrity, our findings also give a mechanistic explanation to these data.

Synthetic Lethality in Pancreatic Cancer: Discovery of a New RAD51-BRCA2 Small Molecule Disruptor That Inhibits Homologous Recombination and Synergizes with Olaparib.

Synthetic lethality is an innovative framework for discovering novel anticancer drug candidates. One example is the use of PARP inhibitors (PARPi) in oncology patients with BRCA mutations. Here, we exploit a new paradigm based on the possibility of triggering synthetic lethality using only small organic molecules (dubbed "fully small-molecule-induced synthetic lethality"). We exploited this paradigm to target pancreatic cancer, one of the major unmet needs in oncology. We discovered a dihydroquinolone pyrazoline-based molecule (35d) that disrupts the RAD51-BRCA2 protein-protein interaction, thus mimicking the effect of BRCA2 mutation. 35d inhibits the homologous recombination in a human pancreatic adenocarcinoma cell line. In addition, it synergizes with olaparib (a PARPi) to trigger synthetic lethality. This strategy aims to widen the use of PARPi in BRCA-competent and olaparib-resistant cancers, making fully small-molecule-induced synthetic lethality an innovative approach toward unmet oncological needs.

Metabolic activation triggered by cAMP in MCF-7 cells generates lethal vulnerability to combined oxamate/etomoxir.

BACKGROUND: Altered energy metabolism is a biochemical fingerprint of cancer cells, widely recognized as one of the "hallmarks of cancer". Cancer cells show highly increased rates of glucose uptake and glycolysis, after which the resulting pyruvate is converted to lactate. The maintenance of this metabolic asset is warranted by lactate dehydrogenase A (LDH-A) and for this reason the development of novel LDH-targeted anticancer therapeutics is underway. However, possible interference in cancer cell metabolism could also arise from cAMP signaling pathway, which could be activated by either oncogenic induction or exogenously, as a result of microenvironment-derived stimuli, increasing cellular cAMP levels. This study aimed at evaluating the impact of activated cAMP signaling pathway on the efficacy of an LDH-targeted anticancer approach. METHODS: We exogenously activated cAMP signaling in MCF-7 human breast cancer cells and explored the metabolic interplay between LDH-A and cAMP pathway. RESULTS: In cAMP-activated cells, we evidenced changes in energy metabolism which reduced their response to LDH inhibition. Interestingly, these experiments also highlighted a potential vulnerability state of treated cells. CONCLUSIONS: cAMP-induced metabolic changes made MCF-7 cells a preferential target of a drug combination treatment which should not affect normal cell viability. GENERAL SIGNIFICANCE: cAMP is a well-recognized second messenger of the pro-inflammatory cascade. The obtained results are relevant in consideration of the crucial role played by inflammation in normal breast cell transformation and in cancer progression. Furthermore, they corroborate the idea of exploiting the metabolic changes observed in cancer cells to obtain a therapeutic advantage.

Rad51/BRCA2 disruptors inhibit homologous recombination and synergize with olaparib in pancreatic cancer cells.

Olaparib is a PARP inhibitor (PARPi). For patients bearing BRCA1 or BRCA2 mutations, olaparib is approved to treat ovarian cancer and in clinical trials to treat breast and pancreatic cancers. In BRCA2-defective patients, PARPi inhibits DNA single-strand break repair, while BRCA2 mutations hamper double-strand break repair. Recently, we identified a series of triazole derivatives that mimic BRCA2 mutations by disrupting the Rad51-BRCA2 interaction and thus double-strand break repair. Here, we have computationally designed, synthesized, and tested over 40 novel derivatives. Additionally, we designed and conducted novel biological assays to characterize how they disrupt the Rad51-BRCA2 interaction and inhibit double-strand break repair. These compounds synergized with olaparib to target pancreatic cancer cells with functional BRCA2. This supports the idea that small organic molecules can mimic genetic mutations to improve the profile of anticancer drugs for precision medicine. Moreover, this paradigm could be exploited in other genetic pathways to discover innovative anticancer targets and drug candidates.

The activation of lactate dehydrogenase induced by mTOR drives neoplastic change in breast epithelial cells.

mTOR kinase and the A isoform of lactate dehydrogenase (LDH-A) are key players controlling the metabolic characteristics of cancer cells. By using cultured human breast cells as a "metabolic tumor" model, we attempted to explore the correlation between these two factors. "Metabolic tumors" are defined as neoplastic conditions frequently associated with features of the metabolic syndrome, such as hyper-insulinemia and hyper-glycemia. MCF-7 cells (a well differentiated carcinoma) and MCF-10A cells (a widely used model for studying normal breast cell transformation) were used in this study. These cells were exposed to known factors triggering mTOR activation. In both treated cultures, we evaluated the link between mTOR kinase activity and the level of LDH expression / function. Furthermore, we elaborated the metabolic changes produced in cells by the mTOR-directed LDH-A up-regulation. Interestingly, we observed that in the non-neoplastic MCF-10A culture, mTOR-directed up-regulation of LDH-A was followed by a reprogramming of cell metabolism, which showed an increased dependence on glycolysis rather than on oxidative reactions. As a consequence, lactate production appeared to be enhanced and cells began to display increased self-renewal and clonogenic power: signals suggestive of neoplastic change. Enhanced clonogenicity of cells was abolished by rapamycin treatment, and furthermore heavily reduced by LDH enzymatic inhibition. These results highlighted a mechanistic link between metabolic alterations and tumorigenesis, whereby suggesting LDH inhibition as a possible chemo-preventive measure to target the metabolic alterations driving neoplastic change.

Impact of potentially inappropriate medications and polypharmacy on 3-month readmission among older patients discharged from acute care hospital: a prospective study.

BACKGROUND: Polypharmacy and potentially inappropriate medications (PIMs) are known to affect several negative outcomes in older patients. However, studies comparatively assessing polypharmacy and PIMs in relation to readmission are distinctively lacking. AIMS: To compare the impact of polypharmacy and PIMs on 3-month readmission among older patients discharged from acute care hospital. METHODS: Our series consisted of 647 patients consecutively enrolled in a multicenter observational study. The outcome of the study was the occurrence of any admission during the 3-month follow-up after discharge. Polypharmacy was defined as use of more than eight medications. PIMs were identified using 2015 version of Beers and Screening Tool of Older Persons Prescriptions (STOPP) criteria. Statistical analysis was performed using logistic regression models. RESULTS: After adjusting for potential confounders, polypharmacy (OR 2.72, 95% CI 1.48-4.99) was found associated with the outcome, while Beers (OR 0.85, 95% CI 0.46-1.56), STOPP (OR 1.60, 95% CI 0.85-3.01), or combined Beers and STOPP violations (OR 0.99, 95% CI 0.57-1.74) were not. The association between polypharmacy and 3-month readmission was confirmed in logistic regression models including Beers (OR 2.88, 95% CI 1.55-5.34), STOPP (OR 2.64, 95% CI 1.43-4.87), or combined Beers and STOPP violations (OR 2.80, 95% CI 1.51-5.21). DISCUSSION: Besides confirming that polypharmacy should be considered as a marker for readmission risk among older patients discharged from acute care hospital, our findings suggest that the association between polypharmacy and 3-month readmission is substantially independent of use of PIMs. CONCLUSIONS: Polypharmacy, but not PIMs was significantly associated with readmission. Hospitalization should always be considered as a clue to individuate unnecessary polypharmacy and to reduce the burden of medications whenever possible.

Synthetic Lethality Triggered by Combining Olaparib with BRCA2-Rad51 Disruptors.

In BRCA2-defective cells, poly(adenosine diphosphate [ADP]-ribose) polymerase inhibitors can trigger synthetic lethality, as two independent DNA-repairing mechanisms are simultaneously impaired. Here, we have pharmacologically induced synthetic lethality, which was triggered by combining two different small organic molecules. When administered with a BRCA2-Rad51 disruptor in nonmutant cells, Olaparib showed anticancer activity comparable to that shown when administered alone in BRCA2-defective cells. This strategy could represent an innovative approach to anticancer drug discovery and could be extended to other synthetic lethality pathways.

LDH inhibition impacts on heat shock response and induces senescence of hepatocellular carcinoma cells.

In normal cells, heat shock response (HSR) is rapidly induced in response to a variety of harmful conditions and represents one of the most efficient defense mechanism. In cancer tissues, constitutive activation converts HSR into a life-threatening process, which plays a major role in helping cell survival and proliferation. Overexpression of heat shock proteins (HSPs) has been widely reported in human cancers and was found to correlate with tumor progression. Hepatocellular carcinoma is one of the conditions in which HSR activation was shown to have the highest clinical significance. Transcription of HSPs is induced by HSF-1, which also activates glycolytic metabolism and increases the expression of LDH-A, the master regulator of the Warburg effect. In this paper, we tried to explore the relationship between HSR and LDH-A. In cultured hepatocellular carcinoma cells, by using two enzyme inhibitors (oxamate and galloflavin), we found that the reduction of LDH-A activity led to decreased level and function of the major HSPs involved in tumorigenesis. Galloflavin (a polyphenol) also inhibited the ATPase activity of two of the examined HSPs. Finally, hindering HSR markedly lowered the alpha-fetoprotein cellular levels and induced senescence. Specific inhibitors of single HSPs are currently under evaluation in different neoplastic diseases. However, one of the effects usually observed during treatment is a compensatory elevation of other HSPs, which decreases treatment efficacy. Our results highlight a connection between LDH and HSR and suggest LDH inhibition as a way to globally impact on this tumor promoting process.

Biocomplexity and Fractality in the Search of Biomarkers of Aging and Pathology: Focus on Mitochondrial DNA and Alzheimer's Disease.

Alzheimer's disease (AD) represents one major health concern for our growing elderly population. It accounts for increasing impairment of cognitive capacity followed by loss of executive function in late stage. AD pathogenesis is multifaceted and difficult to pinpoint, and understanding AD etiology will be critical to effectively diagnose and treat the disease. An interesting hypothesis concerning AD development postulates a cause-effect relationship between accumulation of mitochondrial DNA (mtDNA) mutations and neurodegenerative changes associated with this pathology. Here we propose a computerized method for an easy and fast mtDNA mutations-based characterization of AD. The method has been built taking into account the complexity of living being and fractal properties of many anatomic and physiologic structures, including mtDNA. Dealing with mtDNA mutations as gaps in the nucleotide sequence, fractal lacunarity appears a suitable tool to differentiate between aging and AD. Therefore, Chaos Game Representation method has been used to display DNA fractal properties after adapting the algorithm to visualize also heteroplasmic mutations. Parameter ß from our fractal lacunarity method, based on hyperbola model function, has been measured to quantitatively characterize AD on the basis of mtDNA mutations. Results from this pilot study to develop the method show that fractal lacunarity parameter ß of mtDNA is statistically different in AD patients when compared to age-matched controls. Fractal lacunarity analysis represents a useful tool to analyze mtDNA mutations. Lacunarity parameter ß is able to characterize individual mutation profile of mitochondrial genome and appears a promising index to discriminate between AD and aging.

Lactate dehydrogenase inhibitors can reverse inflammation induced changes in colon cancer cells.

The inflammatory microenvironment is an essential component of neoplastic lesions and can significantly impact on tumor progression. Besides facilitating invasive growth, inflammatory cytokines were also found to reprogram cancer cell metabolism and to induce aerobic glycolysis. Previous studies did not consider the possible contribution played in these changes by lactate dehydrogenase (LDH). The A isoform of LDH (LDH-A) is the master regulator of aerobic glycolysis; it actively reduces pyruvate and causes enhanced lactate levels in tumor tissues. In cancer cells, lactate was recently found to directly increase migration ability; moreover, when released in the microenvironment, it can facilitate matrix remodeling. In this paper, we illustrate that treatment of human colon adenocarcinoma cells with TNF-α and IL-17, two pro-inflammatory cytokines, modifies LDH activity, causing a shift toward the A isoform which results in increased lactate production. At the same time, the two cytokines appeared to induce features of epithelial-mesenchymal transition in the treated cells, such as reduction of E-cadherin levels and increased secretion of metalloproteinases. Noteworthy, oxamate and galloflavin, two inhibitors of LDH activity which reduce lactate production in cells, were found to relieve the inflammation-induced effects. These results suggest LDH-A and/or lactate as common elements at the cross-road between cancer cell metabolism, tumor progression and inflammation. At present, LDH inhibitors suitable for clinical use are actively searched as possible anti-proliferative agents; our data lead to hypothesize for these compounds a wider potential in anticancer treatment.