Betulinic acid may modulate autophagy in renal cell carcinoma cells.

OBJECTIVES: Betulinic acid is pentacyclic triterpenoid known to exert antitumor effects by modulating many cellular pathways in various human malignancies. However, its modulatory role in autophagy in renal cell carcinoma remains unclear. Here, we observed how betulinic acid affects autophagy in renal cell carcinoma cells. METHODS: After treating cells with betulinic acid, we determined the gene expression and protein levels of Beclin-1 and ATG-5 by qPCR and ELISA assay to observe its effects on autophagy. RESULTS: The qPCR results demonstrated that Beclin-1 expression level was low in untreated metastatic renal adenocarcinoma ACHN cells and increased in response to 25 µM and 50 µM betulinic acid treatment. ATG-5 expression level was decreased in primary clear cell renal cell carcinoma CAKI-2 cells treated with 50 µM betulinic acid. In the ELISA assay results, we observed that betulinic acid caused a decrease in Beclin-1 protein level at 25 µM concentration and in ATG-5 protein level at 50 µM concentration in CAKI-2 cells. CONCLUSION: In our preliminarily study, it was concluded that the role of autophagy may differ in renal cell carcinoma cells depending on their origin and that the effects of betulinic acid on autophagy in these cells may vary accordingly (Fig. 4, Ref. 40). Text in PDF www.elis.sk Keywords: betulinic acid, autophagy, kidney, cancer, cell.

The inhibitory effect of betulinic acid on epithelial-mesenchymal transition pathway in renal cell carcinoma.

Renal cancer is the most lethal urological cancer and characterized by high metastasis rate at initial diagnosis and drug resistance to current chemotherapeutics. Betulinic acid is a pentacyclic triterpene with broad biological activity that occurs naturally in variety of plants. Even though the anti-cancer efficacy of betulinic acid have been reported by many studies, the information about the pathways and the molecules which are affected by betulinic acid in renal cancer are limited. Epithelial-mesenchymal transition (EMT) is considered as the initial step of metastasis and contributes to drug resistance of cancer cells. Depending on the role of EMT in cancer progression and drug resistance, targeting EMT may represent an effective strategy in this context. Therefore, we aimed to investigate the anti-metastatic effects of betulinic acid on renal cell carcinoma cells by evaluating two EMT markers, SNAIL-1, and SDC-2. Following the treatment of betulinic acid at determined doses by WST-1 cytotoxicity assay in our previous study, SDC-2 expression level was decreased in both cell lines. Additionally, in correlation with this result, we also found a reduction in SDC-2 and SNAIL-1 protein levels which are measured by ELISA. Furthermore, the migration and invasion capacities were suppressed by betulinic acid treatment in metastatic renal adenocarcinoma ACHN cells. Taken together, our findings indicate that betulinic acid may constitute a potential treatment approach for renal cancer with further investigations.

Effects of betulinic acid on AKT/mTOR pathway in renal cell carcinoma.

OBJECTIVE: Renal cancer is the most lethal among urological cancer. Treatments of renal cell carcinoma (RCC) may be possible by immune checkpoint inhibitors and drug treatment targeting different molecules. We aimed to determine the apoptotic effect of betulinic acid and its effects on expressions of apoptosisassociated genes AKT-1 and mTOR in RCC cells. MATERIAL AND METHODS: In this study, we investigated the apoptotic activity of betulinic acid in CAKI-2 cell line and its effect on AKT-1 and mTOR gene expression levels. In order to do so, following analyses were conducted: WST-1 to identify the toxic effect of betulinic acid, Caspase-3/BCA to detect caspase enzyme activity, Annexin-V and ELISA to determine for apoptotic effect, and finally, real-time PCR for expression levels of AKT-1 and mTOR. RESULTS: Our study showed that different concentrations of betulinic acid induced apoptosis in renal cancer; however, no effect was observed in healthy cells. In gene expression analysis, there was statistically significant decrease in AKT-1 expression level while increasing mTOR expression level. CONCLUSION: We suggested that betulinic acid with its apoptotic effect on RCC line and nontoxic effect on healthy cell line and the effects on AKT/mTOR pathway may be a potential anticancer drug promising for future studies.

Apoptotic effects of acacetin in human colon cancer HT-29 and HCT 116 cells.

AIM: Acacetin is a natural flavone compound, which is found in several plants as Robinia pseudoacacia and is demonstrated to have anticancerogenic activities in many types of cancer (e.g., human nonsmall cell lung cancer, and prostate). Colorectal carcinoma (CRC) is one of the serious health problems and is a complex disease. We intended to find a more effective new candidate for the treatment of colon cancer, and hence, we designed this study to investigate the effects of acacetin on CRC (HT-29, HCT 116) in vitro. METHODS: The study was carried out with the methods that determine for apoptosis (WST-1, Caspase 3/BCA, Annexin V). RESULTS: Acacetin showed antitumor and apoptosis-inducing effects in the CRC cell lines. CONCLUSIONS: Acacetin was effective on CRC cell lines, besides no lethal effect on healthy lung cells (MRC-5).

The apoptotic efficacy of succinic acid on renal cancer cell lines.

Recently, studies on the effects of non-toxic substances on cancer prophylaxis have gained value as an alternative to existing treatment options. Current studies have shown that succinic acid or its derivatives exhibit anticancer activity by inducing apoptosis. We aimed to investigate the anticancer activity of succinic acid on renal cancer for the first time in the literature. The cytotoxic activity of succinic acid on CAKI-2 and ACHN as renal cancer cell lines and MRC-5 as a healthy cell line was determined using the WST-1 cytotoxicity test. Apoptotic activity was measured by Annexin V test and cell death ELISA kit. The results showed that 25 µM and 50 µM doses of succinic acid for 24 h remarkably reduced the cell viability for CAKI-2 cells (89.77% and 90.77%) and ACHN cells (41.57% and 54.54%). Also, no significant effect was observed on the healthy cell line, as we expected. Additionally, administration of succinic acid at same doses resulted in apoptotic activity for ACHN cells (19.1 and 12.7) and CAKI-2 cells (19.85 and 29.55). ELISA results with same doses of succinic acid treatment increased the apoptotic fragment rates by 4.7 and 2.13-fold in CAKI-2 cells, and 32.92, 12.7-fold in ACHN cells. Succinic acid is a focal point for cancer treatments not only for its apoptotic success on cancer cells but also for its capacity to be metabolically active for humans. Our results suggest that succinic acid could be a potential therapeutic agent for individual cancer treatment approaches together with further molecular research.

Levofloxacin might be safe to use for OSCC patients.

Oral squamous cell carcinoma patients are exhausted against the powerful chemotherapies, radiotherapies after the surgery, and their immune system is devastated during the process and antibiotic usage become inescapable. Although prescribing an antibiotic might be fraught for such as drug interaction and undesirable proliferation danger, studies still look for the new ideas such as antibiotic combinations that might be safe to use. The antiproliferative and apoptotic outcomes of levofloxacin with cisplatin combination as well as their single usage were examined with WST-1, Caspase-3/BCA and Annexin V methods on SCC-15 cells and a healthy cell line (MRC-5). 24 h treatment of 50 mM single levofloxacin, 50 mM single cisplatin and 50 mM levofloxacin-cisplatin combination resulted in viability rates of SCC-15 cells as 90%, 67% and 80.8%, respectively. Caspase-3 enzyme activity was enhanced 0.92-fold for single levofloxacin, 13.05-fold for single cisplatin and 9.73-fold for the combination of levofloxacin-cisplatin, the total apoptotic activity of single levofloxacin, single cisplatin and levofloxacin-cisplatin combination were observed as 4.88%, 21.14%, 16.21%, respectively on SCC-15. The apoptotic effect of cisplatin on MRC-5 has been shown to be suppressed when combined with levofloxacin. Considering the cell viability, caspase-3, and apoptotic activity results, it's conclude that the levofloxacin-cisplatin combination was also effective compared to the only cisplatin treatment on OSCC cells. The combination has shown less toxicity for healthy cells than single cisplatin treatment. Therefore, our apoptotic findings suggest that the different dosage combinations are necessary to understand the interaction for the treatment of tongue squamous cell carcinoma.

In Vitro Inhibitory Effect of Succinic Acid on T-Cell Acute Lymphoblastic Leukemia Cell Lines.

BACKGROUND AND AIMS: Although several treatment regimens for T-cell acute lymphoblastic leukemia (T-ALL), trouble is still ongoing that relapse of disease after therapies in both pediatric and adult patients. Hence, the demand for new alternative therapeutics that are antiproliferative for cancer cells but do not harm healthy cells in treatments is increasing day by day. This study aimed to investigate whether succinic acid show anti-proliferative and apoptotic effect of on T-ALL cell lines. METHODS: Time and dose-dependent effects of succinic acid on T-ALL cell lines were determined by using WST-1, caspase-3/ bicinchoninic acid (BCA), and Annexin V-Fluorescein isothiocyanate (FITC) assays. We included the MRC-5 cell line in our study as a healthy control group. RESULTS: Based on our findings, 25 and 50 mmol dosages of succinic acid has shown an apoptotic effect on T-ALL cell lines for 48 h treatment. Also, it has shown that after 48 h exposure of 25 and 50 mmol dosages of succinic acid has no significant cytotoxic effect in healthy MRC-5 cells. Apoptotic activity of succinic acid on CCRF-CEM cell line was caspase-3 dependent but not for MOLT-4. As a consequence, succinic acid was found to effect for T-ALL treatment in vitro and might also enlighten new study fields for different cancer experiments.

G protein gene variants in schizophrenia

Abstract Background Various studies demonstrating enhanced vulnerability to apoptosis may contribute to the pathobiology of schizophrenia. Objective Thus, G proteins may provide an intriguing link between the signal transduction, and apoptotic hypotheses of schizophrenia. In the light of these findings, we investigated whether G protein gene polymorphisms (GNAS1-T393C and GNB3-C825T) accounted for an increased risk of schizophrenia. Methods The present analyses were based on 100 subjects diagnosed with schizophrenia, and on 100 unrelated healthy controls. The genotyping of GNAS1-T393C, and GNB3-C825T gene polymorphisms were performed using the polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP). Results: We demonstrated the positive association of GNB3-C825T gene variants with schizophrenia risk (p: 0.023). In our study, more prevalent CC genotype frequencies were detected in GNB3 in patients compared with the frequencies in the controls. The individuals with GNB3-C825T CC genotype had 2 fold increased risk for schizophrenia (p: 0.011, c2: 6.39, OR:2.14, 95% CI: 1.18-3.90). Discussion Our study results suggested that GNB3-C825T polymorphism might be associated with schizophrenia.

ROS related enzyme levels and its association to molecular signaling pathway in the development of head and neck cancer.

Given the prevalence and annual incidence of cancer, head and neck cancer is affecting more than 600,000 people each year. In this research, it was decided to investigate that which genes are involved and how MPO, NQO1, SOD2 enzyme levels effective to develop of head and neck cancer and for the first time at the tissue level. 35 tumor tissues in all head and neck anatomy and their surrounding tissue (70 in total) were enclosed the research that received surgery. Determination of the apoptosis genes expression levels (Mtch1, Akt1, Caspase3, Caspase9, Bcl2, Mdm2, mTOR) were determined by RT-PCR techniques and the same patients' sample used for ROS associated oxidant-antioxidant system by using MPO, NQO1, SOD2 enzyme levels using ELISA method. According to statistical results, caspase 9 gene was found statistically high expressed in early stage in contrast to late stage (p=0,013). Level of SOD2, NQO1 and MPO was determined and only MPO level was found significantly important on tumor tissues p=0,008).  Specially, our findings for high expression of Cas9 on early stage were thought to be the target for treatment with its well-known initiator role of the apoptosis. Our results suggest that the higher level of MPO in tumor tissues and indicates that it has some role on pathology of head and neck cancers. We believe that, our research will lead the proposal in-vivo studies and will open new areas on therapeutic targets.

An answer to colon cancer treatment by mesenchymal stem cell originated from adipose tissue.

OBJECTIVES: Colon cancer is risen up with its complex mechanism that directly impacts on its treatment as well as its common prevalence. Mesenchymal stem cells (MSCs) have been considered as a therapeutic candidate for conventional disease including cancer. In this research, we have focused on apoptotic effects of adipose tissue-derived MSCs in colon cancer. MATERIALS AND METHODS: MSCs were obtained from adipose tissue and characterized by Flowcytometer using suitable antibodies. MSCs, HT-29, HCT-116, RKO and healthy cell line MRC5 were cultured by different seeding procedure. After cell viability assay, changes in caspase 3 enzyme activity and the level of phosphatidylserine were measured. RESULTS: For cell viability assay, a 48 hr incubation period was chosen to seed all cells together. There was a 1.36-fold decrease in caspase 3 enzyme activity by co-treatment of RKO and MSCs in addition to 2.02-fold decrease in HT-29 and MSCs co-treatment, and 1.103-fold increase in HCT-116 and MSCs. The results demonstrated that HCT-116 led to the highest rate of apoptotic cell death (7.5%) compared with other cells. CONCLUSION: We suggest that MSCs might remain a new treatment option for cancer by its differentiation and repair capacity.

Relation of MPO, MnSOD, NQO1 gene variants in endometrial carcinoma in the line of PCR-RFLP methods.

Reactive oxygen species (ROS) have been shown to be responsible for inducing DNA damage leading to mutagenesis, carcinogenesis, and cell death if the capacity of the protective antioxidant system is impaired. Endometrial carcinoma is the primary cancer type in the female genital system. The enhanced cell lipid peroxidation and impaired antioxidant enzyme activities observed in patients with endometrial cancer indicate the potential for oxidative injury to cells and cell membranes in such patients. The aim of the study was to investigate the possible association between gene variants of superoxide dismutase (SOD), myeloperoxidase (MPO), and NADPH quinone oxido reductase (NQO1), and their possible role in endometrial cancer in Turkish patients. According to results, MPO G+ genotype and AG genotype were significantly increased in patients compared with controls (P<0.001). We suggest that the MPO polymorphism might be a risk for endometrial cancer.

A new target for the treatment of endometrium cancer by succinic acid.

Endometrium cancer is the most common invasive gynecologic malignancy in developed countries. Succinic acid (CO2HCH2-CH2CO2H) is a type of dibasic acid that has uncolored crystal. Succinic acid is used in bakery products and aromatized products. It is naturally found in some vegetables. Succinic acid has no adverse effects because it is metabolized by body cells and has a role in the tricarboxylic acid cycle (TCA) as a cycle media component. The TCA cycle and its enzyme components have some crucial roles for basal cell metabolism. Any mistakes, concentration differences in product, or enzyme deficiencies are important within the cell this cycle. In this proposal project, we aimed to investigate the effect of succinic acid at different doses and at different times in an endometrial cancer cell line. The study was performed using methods that determine for apoptosis (for cytotoxicity, WST-1, for caspase enzyme activity, Caspase 3/BCA; apoptotic determination using flow cytometry; Annexin V; to understand mitochondrial membrane potential; JC-1). The results showed that 5 and 10 µM concentration of succinic acid resulted in apoptosis in endometrium cancer; no such effect was seen in the control cell line, which comprised healthy lung cells.  According to our results, it is thought that succinic acid would be effective for the treatment of endometrial cancer cell lines, thus providing new data for other areas of cancer research.

Are XPD and XPG gene variants related to the mechanism of oral squamous cell carcinoma?

Oral cavity cancers have anatomically a big part of the body system and include several types of cancer. The aim of the study is to investigate the relation between XPG and XPD gene variants in the DNA repair system and oral squamous cell cancers. A total of 111 patients with a pathologic diagnosis of oral squamous cell carcinoma and a control group of 148 healthy volunteers who presented to Istanbul Faculty of Medicine, Department of Otolaryngology & Head and Neck Surgery and Dentistry Faculty were included in the study. Isolation of DNA was achieved using an Invitrogen Purelink Genomic DNA Kit. XPD alleles of Lys751Gln (rs13181) and XPG Asp1104His (rs17655) loci from genomic DNA samples were reproduced using polymerase chain reaction. A statistically significant difference in XPD genotype distribution between control and patient groups was determined (P=0.019). XPD Lys+ was significantly more common in the patient group than in the control group, and a two-fold increased risk for disease was determined. XPD Gln/Gln+ was significantly more common in the control group than in the patient group, and a two-fold decrease in risk for disease was determined (P=0.045). In the other region of the study, there was no statistically significant difference in terms of disease development between XPG genotypes. In conclusion, Lys751Gln polymorphism in the XPD gene could play a role in oral squamous cell development. It is important to increase the numbers of subjects in patient groups and healthy controls in studies to increase the possibility of determining XPD's potential as a molecular risk factor.

Investigation of DNA repair genes in patients with obsessive-compulsive disorder.

BACKGROUND: Obsessive-compulsive disorder (OCD) is a major psychiatric disorder identified mostly by obsessions and compulsions. Molecular genetic and gene-expression studies focused on familial and twin cases have shown a wide variety of variant genes related to OCD. OBJECTIVES: The aim of the study was to investigate DNA repair genes as potential molecular markers in OCD by evaluating the distribution of polymorphisms of DNA repair genes in OCD patients. MATERIAL AND METHODS: The study included 100 case subjects with OCD and 122 unrelated healthy controls. Genotyping of XRCC1, XRCC3, XPD, XPG, APE1 and HOGG1 was performed by polymerase chain reactionrestriction fragment length polymorphism. RESULTS: Significant differences were found for XPD and genotype frequencies. Likewise, the frequency of the XPD Lys+ genotype was significantly increased in the patients as compared to the controls, and carriers of the Lys+ genotype had an increased risk for OCD (p = 0.027). The XPD Lys/Lys genotype frequency was also increased in the patients in comparison to the controls (p < 0.001). XPD Gln+ frequencies were higher in the controls than in the patients, and carriers of the Gln+ genotype showed decreased levels of OCD risk (p < 0.001). XPD Lys/Lys genotype frequency and XPD Gln+ frequency are also significantly associated even after Bonferroni correction (p < 0.008). CONCLUSIONS: The findings suggest that XPD Lys/Lys might play a facilitating role in the development of OCD.