Mechanisms of anti-cancer effects of ascorbate: Cytotoxic activity and epigenetic modulation.

Vitamin C (Vit C or Ascorbate) is essential for many fundamental biochemical processes. Vit C is an essential nutrient with redox functions at normal physiologic concentrations. The main physiologic function of this vitamin is related to its capacity to act as a co-factor for a large family of enzymes, collectively known as Fe and 2-oxoglutarate-dependent dioxygenases. It also modulates epigenetic gene expression through the control of TET enzymes activity. Vit C also has several biological properties allowing to restore the deregulated epigenetic response observed in many tumors. High-dose Vit C has been investigated as a treatment for cancer patients since the 1969. Pharmacologic ascorbate acts as a pro-drug for hydrogen peroxide formation (H2O2) and, through this mechanism, kills cancer cells. To achieve high in vivo concentrations, Ascorbate must be injected by i.v. route. Initial clinical studies of Ascorbate cancer treatment have provided encouraging results, not confirmed in subsequent studies. Recent clinical studies using i.v. injection of high-dose Ascorbate have renewed the interest in the field, showing that significant anti-tumor activity. Pre-clinical studies have led to identify tumors sensitive to Ascorbate that could potentially benefit from this treatment either through an epigenetic modulator effect or through tumor killing by oxidative stress.

High-dose ascorbate and arsenic trioxide selectively kill acute myeloid leukemia and acute promyelocytic leukemia blasts in vitro.

The use of high-dose ascorbate (ASC) for the treatment of human cancer has been attempted several decades ago and has been recently revived by several in vitro and in vivo studies in solid tumors. We tested the cytotoxic effects of ASC, alone or in combination with arsenic trioxide (ATO) in acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL). Leukemic cell lines and primary blasts from AML and APL patients were treated with graded concentrations of ASC, alone or in association with standard concentration (1 µM) of ATO. The ASC/ATO combination killed myeloid blasts, including leukemic CD34+ cells, while sparing CD34+ progenitors obtained from normal cord blood and bone marrow. Actually, approximately one-third (11/36) of primary AML cases were highly sensitive to the ASC/ATO combination. The mechanism of cell killing appeared to be related to increased oxidative stress and overproduction of ROS in a non-quantitative fashion, which resulted in induction of apoptosis. These effects were reverted by the addition of the antioxidant N-Acetyl-Cysteine (NAC). In the APL NB4 model, ASC induced direct degradation of the PML and PML/RARA proteins via caspase activation, while the transcriptional repressor DAXX was recruited in re-constituted PML nuclear bodies. Our findings encourage the design of pilot studies to explore the potential clinical benefit of ASC alone or in combination with ATO in advanced AML and APL.

Cytotoxic effects of high concentrations of sodium ascorbate on human myeloid cell lines.

The effect of high doses of intravenous (sodium) ascorbate (ASC) in the treatment of cancer has been controversial although there is growing evidence that ASC in high (pharmacologic) concentrations induces dose-dependent pro-apoptotic death of tumor cells, in vitro. Very few data are available on the role of ASC in the treatment of acute myeloid leukemia (AML). Ascorbate behaves as an antioxidant at low (physiologic), and as pro-oxidant at pharmacologic, concentrations, and this may account for the differences reported in different experimental settings, when human myeloid cell lines, such as HL60, were treated with ASC. Considering the myeloid origin of HL60 cells, and previous literature reports showing that some cell lines belonging to the myeloid lineage could be sensitive to the pro-apoptotic effects of high concentrations of ASC, we investigated in more details the effects of high doses (0.5 to 7 mM) of ASC in vitro, on a variety of human myeloid cell lines including the following: HL60, U937, NB4, NB4-R4 (retinoic acid [RA]-resistant), NB4/AsR (ATO-resistant) acute promyelocytic leukemia (APL)-derived cell lines, and K562 as well as on normal CD34+ progenitors derived from human cord blood. Our results indicate that all analyzed cell lines including all-trans retinoic acid (ATRA)- and arsenic trioxide (ATO)-resistant ones are highly sensitive to the cytotoxic, pro-oxidant effects of high doses of ASC, with an average 50 % lethal concentration (LC50) of 3 mM, depending on cell type, ASC concentration, and time of exposure. Conversely, high doses of ASC neither did exert significant cytotoxic effects nor impaired the differentiation potential in cord blood (CB) CD34+ normal cells. Since plasma ASC concentrations within the millimolar (mM) range can be easily and safely reached by intravenous administration, we conclude that phase I/II clinical trials using high doses of ASC should be designed for patients with advanced/refractory AML and APL.

Massive anterior chamber involvement after intra-arterial chemotherapy for retinoblastoma: ultrasound biomicroscopy and histopathology.

This case report highlights the usefulness of Ultrasound Biomicroscopy (UBM) in a case of retinoblastoma which showed massive anterior chamber involvement after treatment with intra-arterial chemotherapy. UBM was used to document tumour pseudohypopion, cells in the aqueous humor, implanted clusters of cells on the corneal endothelium, iris nodules, lens capsule deposits and ciliary body invasion. The UBM data, compared with the histopathologic analysis, performed on eye tissue, after enucleation of the affected eye, revealed a significant concordance. UBM may represent an important diagnostic tool in retinoblastoma, when the decision about enucleation of the eye must be made in the absence of histopathologic data.

Successful treatment of macular retinoblastoma with superselective ophthalmic artery infusion of melphalan.

PURPOSE: To report our experience with superselective ophthalmic artery infusion of melphalan (SOAIM) for macular retinoblastoma to obtain tumor control while preserving as much useful vision as possible. METHODS: Five patients with newly diagnosed unilateral retinoblastoma involving the macula were selected within a group of patients eligible for SOAIM as the primary treatment. RESULTS: The mean tumor basal dimension and thickness in this group of five patients with macular retinoblastoma were 11.6 and 12.3 mm, respectively. The stage at diagnosis ranged from II to VB (Reese-Ellsworth) or B to D (International Classification System). Tumor regression with SOAIM was achieved in all cases with regression patterns type I in four cases and III in one case. CONCLUSIONS: SOAIM can be of value in the treatment of macular retinoblastoma. It may allow the salvage of the residual eyesight with a low rate of complications due to the local and systemic toxicity related to chemotherapy.

13q deletion syndrome and retinoblastoma in identical dichorionic diamniotic monozygotic twins.

PURPOSE: To report the case of identical dichorionic diamniotic female twins with unilateral retinoblastoma in 13q deletion syndrome. METHODS: Clinical and ophthalmoscopic evaluation, combination of multiple ligation-dependent probe amplification, array-comparative genomic hybridization analyses, and magnetic resonance imaging were performed. RESULTS: Peculiar facial features, marked hypotonia, gastroesophageal reflux, interatrial septal defect with left to right shunt and light dilatation of right chambers, 5th finger hypoplasia, 3rd-5th toes clinodactyly, 2nd toe overlapped to 3rd toe, and cutis marmorata were found. Ophthalmoscopic evaluation revealed unilateral retinoblastoma in both girls. Magnetic resonance imaging detected corpus callosum hypoplasia in both twins. A 34.4-Mb deletion involving bands 13q13.2-q21.33 and including the RB1 gene was identified in both twins. The deletion was not present in the DNA of their parents and older brother. CONCLUSIONS: Dysmorphic features in children must be always suspicious of 13q deletion syndrome and a short ophthalmoscopic follow-up is necessary to detect the presence of a retinoblastoma.

Adrenaline inhibits the immunological activation of human basophils at pharmacological and ultra-low doses.

BACKGROUND: Adrenaline is known to inhibit the immunological activation of human basophils. The same effect can be obtained with ultra-low doses of histamine. MATERIAL/METHODS: The present investigation was undertaken to verify the effects of ultra-low doses of adrenaline on the immunological activation of human basophils. For this purpose, purified human basophils were isolated from the blood of atopic donors and the response of the basophils to anti-IgE antiserum was evaluated by measuring the release of histamine and the expressions of the activation markers CD63 and CD203c on the basophil surface. RESULTS: The antigenic activation of basophils was significantly decreased by adrenaline at both pharmacological and ultra-low doses. The inhibiting effects of adrenaline were reversed by the beta blocker propranolol at either dose level. CONCLUSIONS: Although seemingly unexplainable by the currently accepted pharmacological principles, this phenomenon, already reported for histamine, deserves further investigation.

Growth of congenital malignant teratoid medulloepithelioma of the ciliary body: a case study.

The purpose of this study was to describe the growth pattern of congenital malignant teratoid medulloepithelioma of the ciliary body by reporting clinical and imaging findings with pathological correlation. An 11-month-old little girl presented with a whitish-pink iris mass in the right eye resulting from a small ciliary body mass consistent with medulloepithelioma at both clinical and computed tomography (CT) findings. At CT, the lesion showed heterogeneous attenuation, without intraocular calcifications. Eleven months later, clinical and ultrabiomicroscopy showed a clear enlargement of the mass, which invaded the pupil. At magnetic resonance imaging (MRI), the lesion showed T1-weighted hyperintensity and T2-weighted slight hypointensity when compared to the vitreous and a notch in the anterolateral aspect of the ipsilateral lens. After intravenous gadolinium administration, the lesion showed intense homogeneous enhancement, and there was leakage of gadolinium in the anterior chamber, resulting from impairment of blood-aqueous barrier. Biopsy revealed a malignant teratoid medulloepithelioma. The eye was then enucleated, and histology confirmed the diagnosis. Systemic chemotherapy and radiotherapy were not performed, since there was no extraocular extension. The 57-month clinical and MRI follow-up did not show disease relapse. This uncommon case displays the natural history of congenital malignant teratoid medulloepithelioma of the ciliary body. While the tumour might have been successfully treated by local excision at diagnosis, the delay in surgical treatment led to tumour overgrowth with consequent need for enucleation. The most important prognostic feature is extraocular extension, which carries a risk of local recurrence, eventually resulting in intracranial extension and/or lymphatic spread.

Retinoblastoma and the genetic theory of cancer: an old paradigm trying to survive to the evidence.

Retinoblastoma (Rb) is considered to represent the prototype of cancer linked to the sequential loss or inactivation of both alleles of a so-called "tumor suppressor gene", the Rb1 gene. The pathogenetic mechanism behind this tumor was first hypothesized by Knudson in 1971 and further confirmed by others who identified the Rb1 gene whose loss or inactivation was claimed to be responsible for the disease. However, after about four decades of continuous research in the field of molecular biology, the evidence behind the role of the Rb1 gene in Rb appears to be seriously flawed in the light of epidemiological, biological, and clinical evidences. This editorial summarizes the inconsistencies on this subject. Nevertheless, the molecular biology establishment still adheres to the biased view of the genetic origin of Rb and other cancers, and hardly any alternative explanations are taken into account.

The retinoblastoma paradigm revisited.

BACKGROUND: Retinoblastoma (Rb) is the most common primary malignant intraocular tumour in childhood. The "two hit" theory, formulated by Knudson in 1971 to explain the variegated clinical expression of the disease, led to the discovery of the so called tumour suppressor genes and the identification of the Rb1 as the prototype of such genes. Mutations of the Rb1 gene are now commonly believed to be the "cause" retinoblastoma, although epidemiological, clinical, and biological evidences argue against it. MATERIAL/METHODS: The Authors have performed a systematic review of available data concerning clinical and diagnostic aspects of retinoblastoma, including molecular genetics. Meta analysis of literature data has been performed in order to validate some of the predictions made by the two hit theory. RESULTS: The following theses are discussed in detail: 1) there is no difference in the age at diagnosis between unilateral and bilateral retinoblastoma; 2) the pathogenetic mechanisms underlying familial, hereditary, and sporadic retinoblastoma are different; 3) bilateral retinoblastoma is not necessarily hereditary; 4) The real incidence of the unilateral phenotype within the familial group is disproportionately higher than that predicted by the "two hit"; 5) retinoblastoma is most probably "caused" by the combination of epigenetic factors and aneuploidy. CONCLUSIONS: Epidemiological, clinical, and more recent biological and genetic evidences, show that the "two hit" theory represents a rather simplistic, outdated, and unreliable model to explain tumour development and clinical evolution of retinoblastoma.

Does the evidence matter in medicine? The retinoblastoma paradigm.

Retinoblastoma (Rb) is the most common intraocular malignant tumour in childhood, with an incidence of 1 in 15,000 live births. Complete information on this rare tumour can be easily accessed through the internet, although many aspect concerning the aetiology and pathogenesis of the disease, are still controversial. The "two hit" theory, formulated in 1971 to explain the variegated clinical expression of the disease, is based on the idea that single gene mutation may determine the development of cancer. However, this view does not take into account the most recent evidences showing the role of aneuploidy and chromosome instability in cancer. Also, a number of other genes and epigenetic mechanisms are involved in the genesis of retinoblastoma. More importantly, the "two hit" theory makes predictions, concerning the age distribution of the tumour, its mode of "transmission" (hereditary retinoblastoma), and its pathogenesis, which are not fulfilled by the clinical reality. Overall, the "two hit" theory represents a rather simplistic and outdated model to explain tumour development and clinical evolution of retinoblastoma.

Hormesis, epitaxy, the structure of liquid water, and the science of homeopathy.

According to the western medical establishment, homeopathy is both "unscientific" and "implausible". A short overview of its history and the methods it uses, however, easily reveals that homeopathy is a true science, fully grounded on the scientific method and on principles, such as, among others, the Arndt-Schultz law, hormesis, and epitaxy, whose plausibility has been clearly and definitely demonstrated in a number of scientific publications and reports. Through a review of the scientific literature, an explanation of the basic principles of homeopathy is proposed based on arguments and evidence of mainstream science to demonstrate that, in spite of the claims of conventional medicine, homeopathy is both scientific and plausible and that there is no reasonable justification for its rejection by the western medical establishment. Hopefully, this hurdle will be overcome by opening academic institutions to homeopathy to enlarge the horizons of medical practice, recover the value of the human relationship with the patient, and through all this, offer the sick a real alternative and the concrete perspective of an improved quality of life.

The growth of a lie and the end of "conventional" medicine.

Throughout its over 200-year history, homeopathy has been proven effective in treating diseases for which conventional medicine has little to offer. However, given its low cost, homeopathy has always represented a serious challenge and a constant threat to the profits of drug companies. Moreover, since drug companies represent the most relevant source of funding for biomedical research worldwide, they are in a privileged position to finance detractive campaigns against homeopathy by manipulating the media as well as academic institutions and the medical establishment. The basic argument against homeopathy is that in some controlled clinical trials (CCTs), comparison with conventional treatments shows that its effects are not superior to those of placebo. Against this thesis we argue that a) CCT methodology cannot be applied to homeopathy, b) misconduct and fraud are common in CCTs, c) adverse drug reactions and side effects show that CCT methodology is deeply flawed, d) an accurate testing of homeopathic remedies requires more sophisticated techniques, e) the placebo effect is no more "plausible" than homeopathy, and its real nature is still unexplained, and f) the placebo effect is nevertheless a "cure" and, as such, worthy of further investigation and analysis. It is concluded that no arguments presently exist against homeopathy and that the recurrent campaigns against it represent the specific interests of the pharmaceutical industry which, in this way, strives to protect its profits from the "threat" of a safer, more effective, and much less expensive treatment modality.

Aberrant methylation of HIN-1 (high in normal-1) is a frequent event in many human malignancies.

HIN-1 (high in normal-1) is a putative cytokine with growth inhibitory activities and is downregulated by aberrant methylation in breast cancers. We studied HIN-1 methylation status in many types of adult and pediatric malignancies and cell lines. We examined the expression of HIN-1 mRNA in 52 cell lines and the promoter methylation status in the cell lines and in over 800 primary tumors representing 17 tumor types using methylation specific PCR. Promoter methylation was observed in 73% of breast cancer, 67% of nonsmall cell lung cancer (NSCLC), 30% of small cell lung cancer (SCLC) and 57% of malignant mesothelioma (MM) cell lines, and methylation was completely correlated with loss of expression. Expression negative cell lines restored HIN-1 expression after treatment with 5-aza-2'-deoxycytidine. Promoter methylation of HIN-1 was found in 90% of retinoblastomas, 73% of Wilms' tumors, 61% of rhabdomyosarcomas, 57% of breast cancers, 52% of prostate cancers, 40% of MMs, 28% of NSCLCs and 27% of lymphomas. Methylation frequencies in colorectal cancers, cervical cancers, bronchial carcinoids, SCLCs, neuroblastomas, osteosarcomas, leukemia, medulloblastomas and bladder cancers were lower (4-21%), while hepatoblastomas lacked methylation. HIN-1 methylation was rarely detected in nonmalignant tissues (8 of 165, 5%). Aberrant methylation of HIN-1 with loss of expression is a common event and may contribute to the pathogenesis of many types of human malignancies.

Aberrant methylation of the HIC1 promoter is a frequent event in specific pediatric neoplasms.

PURPOSE: To determine the role of methylation of HIC1, a candidate tumor suppressor gene on 17p13.3, in various types of pediatric tumors. EXPERIMENTAL DESIGN: We examined the methylation status of the HIC1 promoter by methylation specific PCR in 157 pediatric tumors and 27 nonmalignant tissues. We correlated methylation with mRNA expression by reverse transcription-PCR in eight tumor-derived cell lines. RESULTS: HIC1 methylation was frequent in medulloblastomas (80%, 12 of 15), retinoblastomas (67%, 6 of 9), rhabdomyosarcomas (59%, 13 of 22), germ cell tumors (55%, 6 of 11), and neurouroblastic tumors (36%, 14 of 39); neuroblastomas (43%, 12 of 28); ganglioneuromas (17%, 1 of 6); and ganglioneuroblastomas (20%, 1 of 5). In contrast, a low incidence of methylation was observed in osteosarcomas (17%, 2 of 12), Ewing's tumors (9%, 1 of 11), Wilms' tumors (3%, 1 of 31), and hepatoblastomas (0%, 0 of 7). HIC1 methylation was more frequent in the aggressive alveolar subtype of rhabdomyosarcomas (100%, 8 of 8) than the embryonal subtype (33%, 4 of 12; P < 0.005) and was rare in the nonmalignant tissues examined. Methylation was also demonstrated by sequencing in nine randomly selected tumor samples. Seven of eight pediatric tumor cell lines examined were methylated and showed loss or reduced HIC1 mRNA. Expression was strongly induced in all cell lines by treatment with the demethylating agent 5-aza 2'deoxycytidine. CONCLUSIONS: Our data suggest that aberrant methylation of HIC1 may play a role in the pathogenesis of specific pediatric tumors.

Deregulation of caspase 8 and 10 expression in pediatric tumors and cell lines.

Methylation of the promoter regions of CpG-rich sites in genes is the major mechanism for the silencing of many genes in tumors. Methylation of the key apoptosis-related gene caspase 8 (CASP8) has been reported in some childhood tumors and in neuroendocrine lung tumors. We examined the methylation status of 181 pediatric tumors and found frequent methylation in rhabdomyosarcomas (83%), medulloblastomas (81%), retinoblastomas (59%), and neuroblastomas (52%). Methylation frequencies were low in Wilms' tumors (19%) and absent in hepatoblastomas, acute leukemias, osteosarcomas, Ewing's sarcomas, and ganglioneuromas and in normal tissues. Methylation of CASP8 and the tumor suppressor gene RASSF1A were highly significantly correlated in all tumor types by both the chi(2) and the Fisher's exact tests (P < 0.0001 for both tests). Because the region of the gene examined by us and others is not located in the promoter region and lacks features of a CpG island, we explored the relationship between methylation and gene silencing in detail using 23 pediatric tumor cell lines. Studies included relating the methylation of the region to gene expression at mRNA and protein levels, enzymatic assays of gene function, clonal analysis of PCR amplicons of the region, and exposure to a demethylating agent. These studies indicated that methylation correlated with the loss of gene function in most cases; however, other mechanisms of gene inactivation were present in some cases. Posttranscriptional inactivation of the closely related gene caspase 10 was present in many cell lines. Our results suggest that deregulation of the death receptor pathway to apoptosis is frequent in many types of pediatric tumors and their cell lines.