The non-vesicle cell-free DNA (cfDNA) induces cell transformation associated with horizontal DNA transfer.

BACKGROUND: Cell-free DNA (cfDNA) is a source for liquid biopsy used for cancer diagnosis, therapy selection, and disease monitoring due to its non-invasive nature and ease of extraction. However, cfDNA also participates in cancer development and progression by horizontal transfer. In humans, cfDNA circulates complexed with extracellular vesicles (EV) and macromolecular complexes such as nucleosomes, lipids, and serum proteins. The present study aimed to demonstrate whether cfDNA not associated with EV induces cell transformation and tumorigenesis. METHODS: Supernatant of the SW480 human colon cancer cell line was processed by ultracentrifugation to obtain a soluble fraction (SF) and a fraction associated with EV (EVF). Primary murine embryonic fibroblast cells (NIH3T3) underwent passive transfection with these fractions, and cell proliferation, cell cycle, apoptosis, cell transformation, and tumorigenic assays were performed. Next, cfDNA was analyzed by electronic microscopy, and horizontal transfer was assessed by human mutant KRAS in recipient cells via PCR and recipient cell internalization via fluorescence microscopy. RESULTS: The results showed that the SF but not the EVF of cfDNA induced proliferative and antiapoptotic effects, cell transformation, and tumorigenesis in nude mice, which were reduced by digestion with DNAse I and proteinase K. These effects were associated with horizontal DNA transfer and cfDNA internalization into recipient cells. CONCLUSIONS: The results suggest pro-tumorigenic effects of cfDNA in the SF that can be offset by enzyme treatment. Further exploration of the horizontal tumor progression phenomenon mediated by cfDNA is needed to determine whether its manipulation may play a role in cancer therapy.

Hereditary diffuse gastric cancer (HDGC). An overview.

It is estimated that up to 10% of gastric carcinomas show familial aggregation. In contrast, around 1-3 % (approximately 33,000 yearly) are genuinely hereditary. Hereditary diffuse gastric cancer (HDGC) is a rare malignancy characterized by autosomal dominant inheritance of pathological variants of the CDH1 and CTNNA1 genes encoding the adhesion molecules E-cadherin and α-catenin, respectively. The multifocal nature of the disease and the difficulty of visualizing precursor lesions by endoscopy underscore the need to be aware of this malignancy as surgical prevention can be fully protective. Here, we provide an overview of the main epidemiological, clinical, genetic, and pathological features of HDGC, as well as updated guidelines for its diagnosis, genetic testing, counseling, surveillance, and management. We conclude that HDGC is a rare, highly penetrant disease that is difficult to diagnose and manage, so it is necessary to correctly identify it to offer patients and their families' adequate management following the recommendations of the IGCL. A critical point is identifying a mutation in HDGC families to determine whether unaffected relatives are at risk for cancer.

Pharmacokinetics of hydralazine, an antihypertensive and DNA-demethylating agent, using controlled-release formulations designed for use in dosing schedules based on the acetylator phenotype.

PURPOSE: The antihypertensive hydralazine has recently been repositioned as DNA demethylating for the epigenetic therapy of cancer. As the acetylator phenotype is the key determinant of its plasma levels, the dose of hydralazine needs to be adjusted for the acetylation status of patients. METHODS: The pharmacokinetics of orally administered hydralazine was evaluated in 26 healthy volunteers (13 slow and 13 fast acetylators) after a single dose of 182 mg administered as a controlled-release tablet. Plasma levels of hydralazine were analyzed in 85 cancer patients treated with this formulation at a dose of 83 mg/day and 182 mg/day for slow and fast acetylators, respectively. RESULTS: The C(max) and t(max) of hydralazine for fast acetylators were 208.4 ± 56.9 SD ng/ml and 2.8 ± 2.5 h, respectively. The corresponding results for slow acetylators were 470.4 ± 162.8 ng/ml, and 4.4 ± 3.1 h. Healthy volunteers who were fast acetylators had no clinically significant changes in blood pressure and heart rate or any other side-effect, however, slow acetylators had transient episodes of headache, tachycardia and faintness. Among 85 cancer patients that received either 182 mg or 83 mg of hydralazine daily, according to their acetylator status, the mean concentrations of hydralazine in plasma were 239.1 ng/ml and 259.2 ng/ml for fast and slow acetylators, respectively. These differences were not significantly different, p = 0.3868. CONCLUSIONS: The administration of dose-adjusted controlled-release hydralazine according to the acetylation status of cancer patients yields similar levels of hydralazine.

Epigenetic therapy and cisplatin chemoradiation in FIGO stage IIIB cervical cancer.

INTRODUCTION: This trial aimed to evaluate the safety and efficacy of epigenetic therapy associated with cisplatin chemoradiation in FIGO Stage IIIB patients. METHODS: Hydralazine containing either 182 mg for rapid-, or 83 mg for slow acetylators and magnesium valproate were administered at 30 mg/kg tid. Both drugs were taken until intracavitary therapy was finished. Pelvic external beam radiation and low-dose rate brachytherapy were administered at a total cumulative dose to point A of at least 85 Gy. Weekly cisplatin at 40 mg/m2 was delivered for six cycles. RESULTS: Twenty-two patients were included and 18 (82%) patients completed treatment. Mean dose to point A was 84.6 + 2.2. Median number of cisplatin cycles was 5.5 (range, 1-6). Brachytherapy was delayed for technical reasons; the mean overall treatment time was 11.8 weeks. Grade 3 anemia, leucopenia, neutropenia, and thrombocytopenia were observed in 9%, 45%, 45%, and 9% of patients, respectively. CONCLUSIONS: Hydralazine and valproate are well-tolerated and safe when administered with cisplatin chemoradiation. Unfortunately, the suboptimal administration of brachytherapy for technical reasons in this study, precluded assessing the efficacy of epigenetic therapy. However, the tolerability of this regimen administered concurrent to radiation needs to be further tested.

Mutational analysis of K-ras and Ras protein expression in larynx squamous cell carcinoma.

The ras gene family (H, K and N-ras) encodes the Ras protein, a GTPase-activating protein that regulates several signal transduction pathways including cellular proliferation and differentiation. Mutations in codons 12, 13 and 61 of the ras genes constitute one of the most frequent alterations in human cancer. In the Western Hemisphere, a low frequency of mutations in these genes has been observed in head and neck carcinomas; a higher frequency has been found in countries such as India and Taiwan. Increased protein expression is a relatively frequent event in larynx carcinomas. This study was aimed to evaluate the participation of the k-ras gene and Ras expression in 20 Mexican patients with larynx squamous carcinoma, 2 with dysplasia and 4 with normal mucosa. Samples (of 26 patients) were embedded in paraffin and immunohistochemical analysis was performed for the Ras protein, as well as amplification of the k-ras gene exon 1 (108 bp) by laser capture microdissection. Then, DNA extraction, PCR and sequencing were performed looking for possible mutation in codons 12 and 13. All patients with larynx carcinoma were men, median age 62 years. Eighty-five percent of the patients had risk factors such as smoking and/or alcohol consumption, 25% were in clinical stages I and II, and 75% in stages III and IV; 45% of the patients presented tumor recurrence or persistence. In this study, no mutations were found in codons 12 or 13 of the k-ras gene; however, protein expression was observed in 95% of the samples and a higher expression of the protein was associated with tumor recurrence or persistence, although this was not statistically significant. Unexpectedly, well-differentiated carcinomas and dysplasias presented an increase in protein expression. These results suggest that ras may be involved in early stages of larynx carcinogenesis and may be activated by other mechanisms different from mutations, such as epigenetic events.

Membrane proteins in neoplasic and normal uterine cervix.

Tumor cells exhibit phenotypic and genotypic differences in comparison to normal cells. These differences can be used to identify proteins important for tumor growth and, therefore, potentially used in the diagnosis and follow-up of patients. The objective of this work was to investigate the electrophoretic pattern of cytoplasm membrane proteins from normal and malignant cervix using polyacrylamide-SDS gels. A highly reproducible protein pattern was found in the 29 samples of normal cervix whereas three well-defined patterns of protein bands were observed in the 48 tumor specimens (pattern I: 25%, pattern II: 29.2% and pattern III: 45.8%). A low concentration or absence of high molecular weight proteins was observed (p<0.5) in tumor samples. None of the tumor protein patterns correlated with the clinicopathologic characteristics of patients. Nine out of 11 patients (82%) showing the pattern III had a complete clinical response whereas only 55% (11 out of 20) of those with patterns I and II showed a complete response. However, this difference was non-significant (p=0.1247). In conclusion, we demonstrate that there is a gain and loss of cytoplasmic membrane proteins in tumors, shown as different protein band patterns. These findings could have clinical and biological significance that must be further evaluated.

Matrix metalloproteinases expressed in squamous cell carcinoma of the oral cavity: correlation with clinicopathologic features and neo-adjuvant chemotherapy response.

Secretion of gelatinases A (MMP-2) and B (MMP-9) from 21 tumoral explants of squamous cell carcinoma (SCC) and five samples of normal mucosa of the oral cavity is demonstrated here. The explants were cultured into fetal bovine serum- and phenol red-deprived medium for 48 hours. The gelatinases secreted into the medium were revealed and quantified by zymography and densitometry, respectively. The results showed high medians of the 66 kDa forms of gelatinase A in tumoral explants, in comparison to normal explants: 31.0 vs 5.9 densitometric units (DU) (p <0.01). There was also a relatioship between clinical response to neo-adjuvant chemotherapy and low activity of 66 kDa form of gelatinase A, as well as 84 kDa and 92 kDa forms of gelatinase B. The median of gelatinolysis of the inactive form of gelatinase A (72 kDa form) was higher in those patients who exhibited a complete response to neo-adjuvant chemotherapy. We conclude that gelatinase A is a useful and objective tool to evaluate the response to chemotherapy and the aggressiveness of carcinomas of the oral cavity.

Upregulation of NKG2D ligands and enhanced natural killer cell cytotoxicity by hydralazine and valproate.

Natural killer cells play a role in the immune antitumor response by recognizing and eliminating tumor cells through the engagement of NKG2D receptors with their ligands on target cells. This work aimed to investigate whether epigenetic drugs are able to increase MICA and MICB expression as well as NK cell cytotoxicity. Prostate, colon, breast and cervical cancer cell lines were analyzed for the expression of MICA and MICB at the mRNA and protein levels by RT-PCR, Western blot, flow cytometry and ELISA. The activating mark H3K4m2 at the MICA and MICB promoters was investigated by ChIP assays. Cytotoxicity of NK cells against the target epithelial cancer cells was investigated with the CD107 cytotoxicity assay. The results show that hydralazine and valproic acid not only increase the expression of MICA and MICB ligands of target cells, but also reduce their shedding to the supernatant. This upregulation occurs at the transcriptional level as revealed by increase of the H3K4 activating mark at the promoter of MICA and MICB genes. These effects are paralleled by increased cytotoxicity of NK cells, which was attenuated at different degrees by using blocking antibodies against the NKG2D receptor and ligands. In conclusion, our results demonstrate the ability of hydralazine and valproate to increase the NK activity against epithelial cancer cell lines and suggest that these drugs could reduce the levels of soluble MICA and MICB helping in avoiding tumor-induced suppression of NK cytotoxicity against the tumor.

Pharmacogenetics and pharmacoepigenetics of gemcitabine.

Gemcitabine (2',2'-difluoro 2'deoxycytidine, dFdC) is an analog of cytosine with distinctive pharmacological properties and a wide antitumor-activity spectrum. The pharmacological characteristics of gemcitabine are unique because two main classes of genes are essential for its antitumor effects: membrane transporter protein-coding genes, whose products are responsible for drug intracellular uptake, as well as enzyme-coding genes, which catalyze its activation and inactivation. The study of the pharmacogenetics and pharmacoepigenetics of these two gene classes is greatly required to optimize the drug's therapeutic use in cancer. This review aims to provide an update of genetic and epigenetic bases that may account for interindividual variation in therapeutic outcome exhibited by gemcitabine.

A phase II study of epigenetic therapy with hydralazine and magnesium valproate to overcome chemotherapy resistance in refractory solid tumors.

BACKGROUND: Epigenetic aberrations lead to chemotherapy resistance; hence, their reversal by inhibitors of DNA methylation and histone deacetylases may overcome it. PATIENTS AND METHODS: Phase II, single-arm study of hydralazine and magnesium valproate added to the same schedule of chemotherapy on which patients were progressing. Schedules comprised cisplatin, carboplatin, paclitaxel, vinorelbine, gemcitabine, pemetrexed, topotecan, doxorubicin, cyclophosphamide, and anastrozole. Patients received hydralazine at 182 mg for rapid, or 83 mg for slow, acetylators, and magnesium valproate at 40 mg/kg, beginning a week before chemotherapy. Response, toxicity, DNA methylation, histone deacetylase activity, plasma valproic acid, and hydralazine levels were evaluated. RESULTS: Seventeen patients were evaluable for toxicity and 15 for response. Primary sites included cervix (3), breast (3), lung (1), testis (1), and ovarian (7) carcinomas. A clinical benefit was observed in 12 (80%) patients: four PR, and eight SD. The most significant toxicity was hematologic. Reduction in global DNA methylation, histone deacetylase activity, and promoter demethylation were observed. CONCLUSIONS: The clinical benefit noted with the epigenetic agents hydralazine and valproate in this selected patient population progressing to chemotherapy' and re-challenged with the same chemotherapy schedule after initiating hydralazine and valproate' lends support to the epigenetic-driven tumor-cell chemoresistance hypothesis (ClinicalTrials.gov Identifier: NCT00404508).

Matrix metalloproteinases-2, -3, and -9 secreted by explants of benign and malignant lesions of the uterine cervix.

Elevated expression of matrix metalloproteinases (MMPs) plays a critical role in extracellular matrix (EM) degradation in tumor development and prognosis of different human carcinomas. In cervical carcinoma (Ce Ca), the role of these proteinases in the biological development of this neoplasm is controversial. In the present study, we compared the secretion of MMP-2, MMP-3 and MMP-9 among 29 benign and premalignant cervical lesions (cervicitis and cervical intraepithelial neoplasias) and 46 tumoral explants of Ce Ca. The explants were cultured for 48 h. The gelatinases secreted into conditioned medium were revealed by zymography and quantified by densitometry. The results showed high levels of MMP-3 and MMP-9 in tumoral explants. In contrast, only the pro-MMP-2 was higher in benign cervical lesions, although both active and inactive MMP-2 species are associated with advanced clinical stages in tumoral samples, and only the secretion of MMP-3 was associated with unresponsiveness to radiotherapy. We can conclude that the expression of MMPs is related to the invasive process in Ce Ca and suggest that they may play a role in degradation of the EM during local invasion. In addition, MMP-3 secretion could be a marker of poor prognosis in Ce Ca.

Neuroendocrine marker expression in cervical carcinomas of non-small cell type.

Small-cell carcinomas of the uterine cervix are highly aggressive tumors. Up to 100% of these tumors express at least one neuroendocrine marker such as neuron-specific enolase (NSE), chromogranin A (CgA), and synaptophysin (SYN). In other tumor types such as non-small-cell carcinomas of the lung, colon, and prostate, the presence of these markers has been associated with a better prognosis in some studies, a worsened prognosis in others, or has had no prognostic effect in still other studies. However, little is known about their expression and prognostic significance in the common "non-small-cell" carcinomas of the uterine cervix. The primary tumors of 54 previously untreated patients with histologically confirmed non-small-cell carcinoma of the cervix uteri (squamous carcinoma, adenosquamous carcinoma, and adenocarcinoma) were analyzed by immunohistochemistry for expression of NSE, CgA, and SYN. The expression status was correlated to pathological characteristics and outcome. In addition, the expression of these markers was investigated in cervical carcinoma cell lines. None of the 54 tumors expressed NSE or CgA, although SYN was positive in five tumors (9%) of which four were squamous and one was adenocarcinoma. These five patients relapsed within the first 6 months of follow-up and four have died. Among eight cancer cell lines only one was positive for CgA and another one for SYN. We conclude that the neuroendocrine marker SYN is expressed in a small subset of non-small-cell carcinomas of the cervix and its expression seems to correlate with a poor outcome.