Pilot Study: Evaluation of the efficacy of homeopathic anti-tumor protocols in Ehrlich tumor

Cancer is the second leading cause of death worldwide, with breast cancer one of the tumors with the highest incidence in Brazil, with an estimated 57,120 new cases in 2014. Several forms of treatment and control of cancer symptoms has been studied, among them homeopathy. In the Banerji clinic in India, doctors have used a protocol consisting of 4 homeopathic medicines with relevant results that motivated this study. In our research the Ehrlich tumor was used, a transplantable tumor cell, whose features are similar a breast carcinoma, as discovered in 1905 by Paul Ehrlich. Objective: To analyze the clinical and immunomodulatory Ehrlich tumor in mice treated with the homeopathic medications found in the Banerji Protocol and with thymulin 5CH. Methodology: The project was approved under protocol 158/13 of the Paulista University Ethics Committee. BALB / c mice between 6 and 9 weeks old were used. Each mouse was inoculated with 1x10 7 tumor cells into the peritoneum cavity, to obtain the ascitic form of tumor. Mice were divided in 5 groups (N=6 animals per group), being the following treatments: control treated with sterile water, Conium maculatum 200CH, Thuja occidentalis 200CH, Phytolacca decandra 200CH, Carcinosinum 200CH, every day being 0.1mL/10g body weight. The animals were weighed and the consumption of water and feed was measured daily. Clinical signals and body temperature were also registered daily. The survival of each group was also analyzed. Results: All groups presented reduction of body temperature one day before death. The peak of clinical symptoms in the majority of groups was in the D18, while Carcinosinum treated group had its peak in D29. The observed clinical symptoms were apathy, prostration, cyanosis, priapism, tachydyspnea, genital edema and secretion, decreased general activity, hair bristling and trembling. The control group had loose stools as unique symptom, seen in 20% of mice; the Carcinosinum treated group showed trembling as the sole symptom, that affected 20% of mice. The group treated with Conium maculatum showed higher incidence of clinical symptoms, involving up to 80% of mice, while the group treated with Carcinosinum showed lower incidence of clinical symptoms, affecting no more than 40% of mice, with weight reduction (reduction of ascites) and longer survival, when compared to the other groups. Conclusion: The results of the pilot study directed us to the second stage of the research, in which the histopathological and immunohistochemical observation will be assessed, being Carcinosinum the treatment chosen to this next step.

High-diluted thymulin on Ehrlich tumor growth in mice and the importance of tumor microenvironment

Introduction: The aim of the present study was to describe different biological aspects of Ehrlich tumor in mice, such as body weight evolution, tumor growth rate, histological organization and systemic immune response after treatment with high-diluted thymulin (10-9 M, named 5CH). Methods: Tumor assessment was focused on macro- and microscopic aspects; parameters included occurrence of necrosis, embolism and tumor development, in addition to quantitative analysis of apoptosis (caspase-3), cell proliferation (Ki-67) and angiogenesis (vascular endothelial growth factor - VEGF) by means of specific immunohistochemistry markers. Spleen cell populations were evaluated by flow cytometry analysis. Results: Mice treated with thymulin 5CH exhibited changes in the tumor microenvironment, such as reduced micro-embolism incidence and cytokeratin expression, with increased caspase-3 expression in the tumor cells. These findings indicate some apoptotic activity by the tumor cells induced by the treatment, even though no reduction of the macroscopic tumor mass occurred. No changes in the systemic immune response were detected, as the balance among spleen cell populations remained unchanged. Conclusions: The results indicate that treatment of mice bearing Ehrlich tumor with thymulin 5CH induces some specific changes in the tumor environment. However, it did not influence systemic immunity parameters. Adjuvant use of thymulin 5CH in oncological clinical practice is still a matter of discussion.

Vochysia tucanorum Mart. butanol fraction presents antitumoral activity in vivo and prevents the installation of cachexia in solid Ehrlich tumor model

Background Cancer is a multifactorial disease caused by uncontrolled proliferation of cells. About 50–80% of cancer patients develop cachexia, a complex metabolic syndrome associated with an increase of mortality and morbidity. However, there are no effective therapies in medical clinic for cancer cachexia. Vochysia tucanorum Mart. is a common three of the Brazilian “Cerrado”. The butanolic fraction of V. tucanorum (Fr-BuVt), very rich in triterpenes with various biological activities, might be interesting in being tested in cancer cachexia syndrome. Hence, the present study was undertaken to investigate the antitumoral activity of Fr-BuVt and its potential against cachexia development. Methods Ehrlich tumor was used as model of cancer cachexia. Ascitic Ehrlich tumor cells were collected, processed and inoculated subcutaneously in saline solution (1 × 107/100 μl; ≥95% viability) for the obtention of solid Ehrlich carcinoma. After inoculation, solid Ehrlich carcinoma-bearing mice were treated by 14 consecutive days by gavage with Fr-BuVt (200 mg/kg). Body weight and tumor volume were measure during the treatment period. Tumors were removed, weighed and properly processed to measure the content and phosphorylation levels of key-proteins involved to apoptotic and proliferation process by Western Blot. Muscles and adipose tissues were removed for weighed. Serum was collected to cytokines levels and energetic blood markers measurements. Results The treatment with the Fr-BuVt (200 mg/kg, 14 days) decreased the solid Ehrlich tumor volume and weight besides increased the expression of the pro-apoptotic proteins caspase-3 and BAX, but also decreased the expression of the proteins involved in proliferation NFκB, mTOR and ERK. In addition, our data shows that the administration of Fr-BuVt was able to prevent the installation of cancer cachexia in Ehrlich carcinoma-bearing mice, since prevented the loss of body weight, as well as the loss of muscle and adipose tissue. Moreover, an improvement in some blood parameters such as decrease in cytokines TNF-α and IL-6 levels is observed. Conclusions The study revealed that Fr-BuVt has antitumoral activity and prevent installation of cancer cachexia in Ehrlich model. Therefore, Fr-BuVt may represent an alternative treatment for cancer cachexia.

METHODOLOGICAL ASPECTS OF PHOTODYNAMIC THERAPY OF EHRLICH SOLID CARCINOMA IN BALB/C MOUSE STRAIN WITH VARIOUS TUMOR LOCALIZATION

Background. Photodynamic Therapy is one of the treatment methods used in modern oncology. Evaluation of the efficacy in vivo of photosensitizers on tumor models is generally accepted, but the photodynamic therapy technique in mice is not without drawbacks.The purpose of the study was evaluation of the efficacy of photodynamic therapy in mice with Ehrlich tumor model after subcutaneous and intracutaneous injection of tumor cells.Material and Methods. The study was conducted on BAL B/C mice of both sexes. Fotoditazin® and Radachlorin® were used as photosensitizers. For photoactivation, the Alod laser apparatus with a wavelength of 662 nm was used.Results. A comparison of photodynamic therapy with subcutaneous and intracutaneous localization of Ehrlich tumor was performed. It was shown that depending on the location and depth of inoculation of Ehrlich tumor, the pharmacokinetics (both the fluorescence intensity over time and the contrast ratio of the tumor/surrounding tissue) and pharmacodynamics (tumor growth inhibition, survival) of photosensitizers are significantly different. Higher contrast of the tumor/surrounding tissue is observed with intracutaneous localization of the tumor.Conclusion. A model with intracutaneous localization of Ehrlich tumor can be recommended for a primary assessment of efficacy; it allows the use of fewer animals in the experiment. When planning experiments to study photosensitizers and evaluating their results, the advantages and disadvantages of different methods for modeling tumors in mice should be taken into account.

Evaluation of in vitro Cytotoxic Effects of Especifico Pessoa Phytotherapic Tincture on Ehrlich Tumor Cells and Mice Spleen Cells

Especifico Pessoa (EP) is traditionally used for the treatment of snakebite envenoming. The traditional use of EP and its properties have been reported. In this study, we evaluated the in vitro cytotoxic effects of EP on Ehrlich tumor and mice spleen cells. Cytotoxicity assay was carried out by using Trypan blue exclusion method. Spleen cell suspension was prepared (n=2) with RPMI medium and tumor cell suspension was prepared from ascitic fluid of Ehrlich tumor-bearing mice (n=1); both the suspensions contained 4 x 106 cells mL-1. Pure EP or EP diluted in RPMI (1:2; 1:4) was used. The results were expressed as percentage of cell viability and demonstrate that EP is toxic to Ehrlich cells at all concentrations (Control: 96.42 ± 3.40; Pure: 1.55 ± 2.91; 1:2: 4.85 ± 5.04; 1:4: 13.39 ± 5.08), but nontoxic to spleen cells in at the lowest dilution (Control: 72.86 ± 13.79; Pure: 13.52 ± 6,36; 1:2: 41.36 ± 13.51; 1:4: 56.59 ± 8.62). Therefore, the results demonstrate that EP has cytotoxic effects, depending on the dose and the cell line evaluated.