Effect of Limonene on Cancer Development in Rodent Models: A Systematic Review

Cancer is a major health issue and one of the leading causes of death worldwide. Many natural compounds, e.g., lycopene, curcumin, resveratrol, etc., have been shown to inhibit the growth of cancer cells. Similarly, limonene, a major active component in citrus essential oils and widely used flavoring additive, has demonstrated anticarcinogenic effects in both cell and animal studies. This systematic review aimed to evaluate the anticarcinogenic effects of limonene and its potential underlying mechanisms. Eight peer-reviewed articles published in English between 2000 and 2020 were identified after screening using MEDLINE, Academic Search Premier, and CINAHL plus. All 8 studies showed an effect of limonene on reducing tumor burden, resulting in either decreased size, number, weight, or multiplicities of tumors. Limonene treatment extended the latency and survival periods in 2 studies yet did not reduce tumor incidence rate in another study. Limonene was shown to promote cell apoptosis in 4 studies that examined either the apoptosis index or apoptosis related gene/protein expressions. Two studies tried to explain the cancer preventive mechanisms of limonene and found limonene could restore the antioxidant capacity or immune functions that were impaired by cancer. These results supported the potential applicability of limonene on inhibiting cancer development, yet the real-world applicability on human requires more research and evaluation through clinical studies.Systematic Review Registration: PROSPERO, identifier: CRD42020168387.

Study on the Relationship between Cardiomyocyte Apoptosis and Left Ventricular Function in Spontaneously Hypertensive Rats

At present, hypertension is a relatively common cardiovascular disease. It not only affects the normal operation of target organs such as the heart and kidneys, but also causes cardiovascular and cerebrovascular diseases, which can lead to death. The apoptosis of cardiomyocytes is widespread in the cardiovascular system and is closely related to vascular diseases. Therefore, the purpose of this article is to explore the relationship between changes in left ventricular function, myocardial multidimensional strain and interstitial fibrosis in spontaneously hypertensive rats (SHR) during cardiomyocyte apoptosis. Whether the research is consistent in terms of order, as the age of hypertensive rats increases, whether there is a close connection between myocardial cell apoptosis and the structure and function of the left heart. The method in this article is to use the method of experimental comparison to randomly group 60 experimental mice and observe the changes of various indicators of rats of different ages, from 12 weeks to 84 weeks. The observations include left ventricular mass index (LVMI), left ventricular ejection fraction (LVEF), left ventricular short axis shortening rate (FS), LVEDP, LV+dp/dtmax, LV-dp/dtmax. At the same time, using the TUNEL labeling method, the left ventricular myocardial tissue was sliced, and the apoptosis index of subendocardial and subepicardial myocardial cells was calculated. Then 3 groups were randomly selected from the experimental group, and Western blotting was used to quantitatively detect apoptosis-related the expression of the proteins Bcl-2, Bax, and Fas were compared between the groups. After analysis and determination, it can be found that the apoptosis index of cardiomyocytes is positively correlated with LVMI, CVF, and PVCA (r is 0.83, 0.89, 0.72, respectively, p=0.00). Corresponding conclusions are drawn from the comparison of data. As hypertensive rats grow older, the apoptotic index of cardiomyocytes will continue to increase, and when the myocardial hypertrophy is severe to heart failure, the apoptotic index of cardiomyocytes will increase significantly. This shows that the increase in cardiomyocyte apoptosis is closely related to left heart remodeling, the development of myocardial fibrosis and overall cardiac dysfunction.

Structural and functional state of Heren carcinoma after local fractional X-irradiation and irradiation combined with meloxicam

Background. One of the most important problems of oncology is the overcoming of therapeutic resistance of tumors, which occurs in particular due to increased levels of the enzyme cyclooxygenase 2 (COX-2). It is known that the growth of COX-2 and the product of its activity, prostaglandin-E2 in cancer, promotes such processes in the body as tumor growth, stimulation of proliferation, induction of cancer stem cells, inhibition of apoptosis, activation of angiogenesis, invasion, metastasis and development of chemoresistance. The use of COX-2 inhibitors, which are nonsteroidal anti-inflammatory drugs (NSAIDs), significantly limits these processes and improves survival and mortality in cancer patients, and in combination with chemotherapeutics eliminates the resistance they cause. Purpose – study of the structural and functional state of Guerin’s carcinoma cells after the combined use of nonsteroidal anti-inflammatory drug meloxicam and local X-irradiation in total doses of 1.0 and 10 Gy. Materials and methods. On 33 rats with inoculated Guerin’s carcinoma, the ultrastructure of tumor cells (TC) was studied using standard methods of electron microscopy 24 hours after the combined use of the meloxicam drug at a dose of 0.2 mg per 1 kg of body weight one day before the first and 2 hours before the second session fractional local X-irradiation in total doses of 1 and 10 Gy (twice daily at 0.5 and 5.0 Gy, respectively. The mitotic index (the number of cells in the state of mitosis per 100 TC,%), the apoptosis index (the number of cells in the state of apoptotic death per 100 TC,%) and the frequency of TC with small nuclei (%). Results. It was found that irradiation of Guerin’s tumor in a total dose of 10 Gy causes disturbances in the ultrastructure associated with damage to the nuclear apparatus of the TC. Pleiomorphism of the nuclei, the appearance of binucleated cells and micronuclei, a significant decrease in mitotic activity and a slight increase in the apoptosis index are observed. Stimulation of the functional activity of macrophages is also noted. Under irradiation in a total dose of 1 Gy, such effects are less pronounced or completely absent, such as, for example, the processes of phagocytosis. The frequency index of TC with small nuclei is equally reliably increased at both radiation doses. The administration of the drug meloxicam leads to a significant decrease in mitotic activity and an increase in the frequency of small cells, while the ultrastructural picture of the tumor remains almost unchanged. With the combined action of the drug and radiation in both doses, violations of the fine structure of the OC are identical to those found during irradiation. At the same time, the mitotic index in the group with the combined effect of the drug and radiation at a dose of 10 Gy is significantly lower than with only irradiation.In addition, at both doses, the frequency of small forms of PC significantly increases in comparison with the indicators of both the intact control group and the corresponding irradiation groups. Only in combination with radiation does meloxicam reliably stimulate apoptosis, while in other groups its index remains at the level of control values. The relationship was confirmed, which was constantly revealed in all experimental groups, between a decrease in the level of the mitotic index and an increase in the frequency of TC with small nuclei in Guerin’s carcinoma. An inverse correlation was found between these indicators (r = 0.80, P = 0.05). Conclusions. The combined action of the drug and irradiation significantly increases the effectiveness of both therapeutic factors due to the property of meloxicam to reliably inhibit proliferative activity and promote post-radiation development of apoptosis in tumor tissue. The presence of a correlation between the mitotic index and the frequency of cells with small nuclei in Guerin’s tumor may indicate the relationship between cell growth and division. Under the combined action of both investigated factors, changes in the tumor ultrastructure are mainly caused by irradiation. The administration of meloxicam increases the efficiency of the combined use of both therapeutic agents due to its ability to reliably inhibit proliferative activity and promote post-radiation development of apoptosis in tumor tissue. The presence of a correlation dependence between the mitotic index and the frequency of cells with small nuclei in Guerin’s tumor may indicate the relationship between the processes of cell growth and division.

Quercetin Improves the Apoptotic Index and Oxidative Stress in Post-Thaw Dog Sperm

This study was conducted to investigate if quercetin (QRN) may ameliorate apoptosis and oxidative stress in post-thaw dog sperm. Herein, we evaluated the post-thaw apoptosis and oxidative stress after treatment with QRN (control, 25, 50, and 100 µM) in freezing of dog semen. The oxidative stress index was significantly affected (p<0.05) between the various concentrations of QRN and the control (17.56 ± 1.02, 7.54 ± 0.48, 5.66 ±0.80, and 10.41 ± 0.69), respectively. The apoptosis index was 9.1 ± 1.34, 6.66 ± 0.58, 6.77 ± 0.66, and 5.38 ± 0.86 in the control, and 25, 50, and 100 µM QRN treatment groups, respectively (p< 0.05). The effects of ameliorated cryo-induced damage by QRN on post-thaw sperm quality were also observed through improved structural and functional tests. Sperm treated with 50 µM QRN showed significantly higher motility (51.8 ± 2.1% vs. 43.1 ± 1.4%, P < 0.05), survival rates (46.9 ± 0.7% vs. 43.9 ± 0.4%, P < 0.05), and mucus penetration than control group, respectively. Results demonstrate that supplementing freezing buffer with 50 µM QRN reduced oxidative damage and improved the quality of post-thaw dog sperm.

Study of the Effects of Homeopathic Medicine Carcinosinum on Mammary Adenocarcinoma (4T1 cells) in vitro.

Background: There are few published researches about the exclusive use of Carsinosinum in several potencies to treat cancer. The name Carcinosinum refers to any homeopathic preparation of epithelial cancerous tissues and is especially indicated when there are any hereditary and familial antecedents of cancer, tuberculosis, diabetes, pernicious anemia or a combination of two or more of these diseases. Homeopathic complexes which include Conium Maculatum, Sabal Serrulata, Thuja Occidentalis and Carcinosinum can reduce in 23% the incidence of prostate cancer in vivo and in 38% the tumor volume, compared to untreated groups. Another in vivo study revealed reduction of symptoms and increase of survival time in mice bearing Ehrlich ascitic carcinoma, after treatment with Carcinosinum 200cH. In vitro, Carcinosinum 200cH can increase the expression of the pro-apoptotic gene p53. However, mice treated with Carcinosinum 6cH had the highest percentage and diversity of symptoms compared to other treatments, which demonstrate the importance of homeopathic potency in pro or anti-carcinogenic action. Considering that the literature on this subject is still rare and focused on genotypic and clinical effects, the present study was proposed, with the aim of identifying the possible phenotypic changes, including viability, HER-2 expression and metastatic skills, using 4T1 cells in vitro as a model, after treatment with Carcinosinum in different homeopathic working dilutions (12cH; 30cH; 200cH), prepared mechanically (Denise Machine, Autic®) in our laboratory using sterile pure water, from a commercial matrix (HN Cristiano, São Paulo, Brazil) stocked in 70% hydro-alcoholic solution. The final dilutions were inserted in the culture medium in a volume equal to 10%, at the time of cell seeding. The same succussioned vehicle used to prepare the medicines (70% hydro-alcoholic solution), from the same batch and diluted 1:100 in sterile pure water, was used as control. All treated cells were cultivated in bottles of 25ml with cell density of 5 x 105 cells / ml and, after 24 hours of treatment, they were analyzed for the apoptosis index using the Annexin V kit and measured by the Countess® system. The morphology of the 4T1 cells was monitored by staining fixed cell smears with hematoxylin-eosin method. The samples were evaluated in quadruplicate and the data were analyzed by one-way ANOVA. The results obtained up to now show that the treatment with Carcinosinum 12cH produced a different pattern of cell death compared to the other treatments, with significant reduction in apoptosis index (one-way ANOVA, p=0.01) and clear hydropic degeneration phenotypic pattern. The analysis of HER-2 expression and metastatic skill will be the next step of this research.

Effects of Phytolacca decandra on the viability of murine breast adenocarcinoma (4T1 cells) in vitro

Background: Comparative studies in cancer patients using conventional and alternative therapy have demonstrated that Phytolacca decandra in homeopathic potencies increases survival and improves quality of life of patients bearing breast cancer. In vitro studies show the induction of apoptosis pathways in MCF-7, a human breast cancer cells lineage, after treatment with Phytolacca decandra in different homeopathic dilutions (from 30C to 10M). Recently, we observed significant growth reduction of Ehrlich carcinoma in mice treated with Phytolacca decandra 30cH. Aims: To evaluate Phytolacca decandra effect in different homeopathic dilutions on the phenotypic features, apoptosis index, and cell morphology of 4T1 cells (murine carcinoma cell lineage) in vitro. Method: The potencies 6, 12, 30 and 200 CH prepared in sterile pure water were studied. Dynamized sterile pure water was used as control. The cytotoxicity was evaluated after different cell treatments in culture bottles (25ml) with the homeopathic medicines (equal to 10% of total medium volume). Cells were cultured in a cell density of 5 x 105 cells / ml, treated with the respective potency and, after 24 hours, analyzed for the apoptosis index using Annexin V kit and measured using the Countess® System. The morphology of the 4T1 cells was monitored by staining fixed cell smears with hematoxylin-eosin method. Cells were previously adhered to a glass cover slip and fixed with absolute methanol. The samples were evaluated in quadruplicate and the data were analyzed by one-way ANOVA. Results and discussion: The results obtained up to now show that the treatment with Phytolacca decandra 200cH induced increase of apoptosis index in relation to the control. Moreover, morphological changes were observed in the respective cell smears: the presence of multinucleated cells, some of them presenting up to 8 nuclei and the increase of eosinophilic staining pattern of cytoplasm, even in mononucleated cells. Conclusion: The increase in apoptosis index reproduced the results described in the literature with other cell lineages, but the changes in morphology still deserve further evaluation.

Effect of Isolated Serum from Breast Cancer Patients with Pectoral Nerves Block on Breast Cancer Cell Line (MDA-MB-231) Apoptosis Index

Background: Breast cancer (BC) is the most frequent cause of cancer death in women. The thoracic pectoral nerve (PECS) block has been described as the gold standard analgesic modality for BC surgery. It has been previously reported that PECS is associated with decreased BC recurrence post-mastectomy. Although several anesthetic drugs and techniques are used in surgical oncology, their effects on the behavior of cancer cells are yet to be known and the key question of whether the anesthetic technique affects cancer outcome remains unresolved. Objectives: Since anesthetic drugs and techniques and post-operative pain may affect BC recurrence, this study aimed to determine whether the anesthetic choice and technique, PECS II block, affects in vitro apoptosis of the MDA-MB-231 BC cell line. Methods: Twenty-two female BC patients, 20 to 75-years-old, with the same pathologic grades were included in this study. The patients were randomly divided into two groups. The first group received propofol general anesthesia (PGA) associated with PECS and the second group received standard PGA. Blood was sampled pre and post-operation from all patients. The sera were isolated and then exposed to the MDA-MB-231 human BC cell line. The mean percentage of apoptosis indices was analyzed by flow cytometry using Annexin V-fluorescein isothiocyanate 24 hours after treatment with patients’ sera. Results: A significant decrease was seen in the mean viability percentage of BC cell line in the PECS group, besides a significant increase in the mean percentage of necrosis and late apoptosis indices compared to the control group after exposure to sera collected from patients post-operation. Intra-group analysis of the control group showed that the exposure of the tumoral cell to post-operation sera resulted in a significant increase in the mean percentage of necrosis and late apoptosis index compared to pre-operation sera exposure. In the PECS group, the exposure of the tumoral cell to post-operation sera resulted in a significant increase in the mean percentage of cell viability and late apoptosis index compared to pre-operation sera exposure. Conclusions: In conclusion, anesthesia and BC surgery may induce apoptosis indices in the MDA-MB-231 human BC cell line. We also found that sera collected from PECS II block patients with BC could induce more apoptosis in the MDA-MB-231 cell line compared to collected sera from systemic analgesia alone after BC surgery.

Therapeutic effect of YSY01 on osteoarthritis in rabbit: involvement of suppressing signaling pathway of NF-kB and MMP-9

IntroductionIt has been reported that the NF-κB and MMP-9 signaling pathways were involved in the pathogenesis of osteoarthritis (OA), while the treatment of OA by YSY01 could inhibit the proteasome activity. Therefore, we aimed to study the therapeutic effect of YSY01A treatment on OA via interfering with expression of NF-κB and MMP-9.Material and methodsWestern blot analysis and immunohistochemistry (IHC) assays were used to measure the expression of NF-κB and MMP-9 in animal models established via SH treatment or cellular models established via sodium nitroprusside (SNP) treatment. MTT assay and flow cytometry analysis were performed to observe the effect of YSY01 treatment on cell viability and apoptosis.ResultsThe decreased expression of NF-κB and MMP-9 was observed in OA rabbits and cells treated by YSY01A, thus indicating the inhibitory effect of YSY01A on NF-κB and MMP-9 expression. And YSY01A exhibited a positive therapeutic effect on OA both in vivo and in vitro by inhibiting the expression of NF-κB and MMP-9. Meanwhile, YSY01A treatment could increase cell viability to a certain degree and decrease the apoptosis index, which suggested the application of YSY01A in OA therapy.ConclusionsThe levels of NF-κB and MMP-9 which were associated with aggravated apoptosis were up-regulated in OA models in vivo and in vitro. YSY01A treatment could down-regulate the expression of NF-κB and MMP-9 and inhibit cell apoptosis, thus reducing the severity of OA.