Induction Of Apoptotic Events Research Articles

6]-Shogaol Induces Apoptosis of Murine Bladder Cancer Cells.

Bladder cancer is considered one of the most aggressive neoplasms due to its recurrence and progression profile, and even with the improvement in diagnosis and treatment methods, the mortality rate has not shown a declining trend in recent decades. From this perspective, the search and development of more effective and safer therapeutic alternatives are necessary. Phytochemicals are excellent sources of active principles with therapeutic potential. [6]-Shogaol is a phenolic compound extracted from the ginger rhizomes that has shown antitumor effects in a wide variety of cancer models. However, there is no record in the literature of studies reporting these effects in models of bladder cancer. Thus, this study aimed to investigate the in vitro cytotoxic and pro-apoptotic potential of [6]-Shogaol against murine bladder cancer urothelial cells (MB49). The cytotoxic effects of [6]-Shogaol on cell viability (MTT method), cell morphology (light microscopy), alteration of proliferative processes (clonogenic assay), oxidative stress pathway (levels of reactive oxygen species) and the induction of apoptotic events (flow cytometry and high-resolution epifluorescence imaging) were evaluated in murine urothelial bladder cancer cell lines (MB49), relative to non-tumor murine fibroblasts (L929). The results showed that [6]-Shogaol was able to induce concentration-dependent cytotoxic effects, which compromised cell viability, exhibiting an inhibitory concentration of 50% of cells (IC50) of 146.8 µM for MB49 tumor cells and 236.0 µM for L929 non-tumor fibroblasts. In addition to inhibiting and altering the proliferative processes if colony formation, it presented pro-apoptotic activity identified through a quantitative analysis and the observation of apoptotic phenotypes, events apparently mediated by the induction of nuclear fragmentation. The data presented suggest that [6]-Shogaol has a higher concentration-dependent cytotoxic and apoptosis-inducing potential in MB49 cells than in L929 fibroblasts. These results may contribute to the development of therapeutic alternatives for bladder cancer.

Ethanolic extract from leaves of tithonia diversifolia induces apoptosis in HCT-116 cells through oxidative stress

ABSTRACT Tithonia diversifolia is a perennial bushy plant found in South America with significant ethnopharmacological importance as an antimalarial, antidiabetic, antibacterial, and anticancer agent. The aim of the present study was to determine the cytotoxicity of the ethanolic extract from leaves of T. diversifolia (TdE) on human cancer cell lines (HCT-116, SNB-19, NCIH-460 and MCF-7), as well as the mechanism of action involved in cell death and cellular modulation of oxidative stress. The TdE exhibited significant activity with IC50 values ranging from 7.12 to 38.41 μg/ml, with HCT-116 being the most sensitive cell line. Subsequent experiments were conducted with HCT-116 cell line. TdE decreased the number of viable cells, followed by induction of apoptotic events, increase in mitochondrial membrane permeabilization, and enhanced G2/M phase of the cell cycle. Pro-oxidative effects including elevated acidic vesicular organelle formation, lipid peroxidation, and nitric oxide by-products, as well as reduced levels of intracellular glutathione and reactive oxygen species production were also observed following incubation with TdE, which may lead to DNA damage followed by apoptotic cell death. These results demonstrate the potential of TdE ethanolic leaf extraction for biological activity and enhance the importance of continuing to study natural sources of plants for the development of anticancer agents.

Temozolomide alleviates breast carcinoma via the inhibition of EGFR/ERK/ MMP-1 pathway with induction of apoptotic events.

To evaluate the chemotherapeutic activity of temozolomide counter to mammary carcinoma. In-vitro anticancer activity has been conducted on MCF7 cells, and mammary carcinoma has been induced in Wistar rats by introduction of 7, 12-Dimethylbenz(a)anthracene (DMBA), which was sustained for 24 weeks. Histopathology, immunohistochemistry, cell proliferation study and apoptosis assay via TUNEL method was conducted to evaluate an antineoplastic activity of temozolomide in rat breast tissue. IC50 value of temozolomide in MCF7 cell has been obtained as 103μM, which demonstrated an initiation of apoptosis. The temozolomide treatment facilitated cell cycle arrest in G2/M and S phase dose dependently. The treatment with temozolomide suggested decrease of the hyperplastic abrasions and renovation of the typical histological features of mammary tissue. Moreover, temozolomide therapy caused the downregulation of epidermal growth factor receptor, extracellular signal-regulated kinase, and metalloproteinase-1 expression and upstream of p53 and caspase-3 proliferation to indicate an initiation of apoptotic events. The occurrence of mammary carcinoma has been significantly decreased by activation of apoptotic pathway and abrogation of cellular propagation that allowable for developing a suitable mechanistic pathway of temozolomide in order to facilitate chemotherapeutic approach.

IBtkα Activates the β-Catenin-Dependent Transcription of MYC through Ubiquitylation and Proteasomal Degradation of GSK3β in Cancerous B Cells.

The IBTK gene encodes the IBtkα protein that is a substrate receptor of E3 ubiquitin ligase, Cullin 3. We have previously reported the pro-tumorigenic activity of Ibtk in MYC-dependent B-lymphomagenesis observed in Eμ-myc transgenic mice. Here, we provide mechanistic evidence of the functional interplay between IBtkα and MYC. We show that IBtkα, albeit indirectly, activates the β-catenin-dependent transcription of the MYC gene. Of course, IBtkα associates with GSK3β and promotes its ubiquitylation, which is associated with proteasomal degradation. This event increases the protein level of β-catenin, a substrate of GSK3β, and results in the transcriptional activation of the MYC and CCND1 target genes of β-catenin, which are involved in the control of cell division and apoptosis. In particular, we found that in Burkitt’s lymphoma cells, IBtkα silencing triggered the downregulation of both MYC mRNA and protein expression, as well as a strong decrease of cell survival, mainly through the induction of apoptotic events, as assessed by using flow cytometry-based cell cycle and apoptosis analysis. Collectively, our results shed further light on the complex puzzle of IBtkα interactome and highlight IBtkα as a potential novel therapeutic target to be employed in the strategy for personalized therapy of B cell lymphoma.

Alternariol alleviates breast carcinoma by inhibiting cellular proliferation correlated with increased apoptotic events in rats

The breast cancer is most frequently diagnosed cancer in the women worldwide. Our study investigated the anticancer effect of alternariol, a secondary metabolite originated from endophytic fungi, against DMBA induced breast carcinoma on Wistar rats. The toxicity study investigated the LD50 and the subsequent doses of alternariol for the carcinogenic study. The breast cancer was developed in rats via induction of DMBA (5 mg/kg, i.v.) and the carcinogenic study was continued for 24 weeks. The induction of breast cancer and the chemotherapeutic effect of alternariol were assessed through histopathological analysis of rat mammary tissue, followed by immunohistochemical analysis, cell proliferation assay and apoptosis assay by TUNEL method. The result showed that alternariol therapy decreased the hyperplastic lesions of mammary tissue and restored the normal histopathological characteristics of breast tissue. Furthermore, alternariol treatment downregulated the expression of carcinogenic markers such as PI3K and Akt increased the expression of apoptotic markers including p53, caspase-3 and Bax. Alternariol therapy also decreased the cellular proliferation and enhanced the apoptotic events. In conclusion, the breast cancer progression was significantly reduced via induction of apoptotic events and inhibition of cell propagation which allowed constructing of suitable mechanism for alternariol mediated chemotherapeutic approach.

Preventive effect of D-carvone during DMBA induced mouse skin tumorigenesis by modulating xenobiotic metabolism and induction of apoptotic events

The structural integrity and excellent immune system of the skin makes it a protective covering, inspite of its exposure to hazardous compounds. In the present study, the chemopreventive efficacy of D-carvone was studied in 7, 12-dimethylbenz[a]anthracene (DMBA) induced skin carcinogenesis. DMBA (25 μg in 0.1 m L−1acetone) was used to induce skin cancer in Swiss albino mice. Animals were randomly divided into six groups of six animals in each. Different concentrations of D-carvone (10, 20, 30 mg/kg body weight) were used to assess its anticancer effect. Tumor incidence, tumor volume, tumor burden, histological examination and levels of phase I and phase II detoxification agents were analyzed in experimental animals. Further, expression of p53 and various apoptotic proteins including- Bcl-2, Bax was analyzed using immunohistochemistry and enzymatic expression of apoptotic proteins caspase-3 and caspase-9 was carried out by using ELISA. We observed 100% tumor incidence in DMBA-painted animals and our results showed that D-carvone at 20 mg dose significantly prevents skin carcinogenesis. Our results also showed decreased levels of phase I enzymes (Cyt P450 and-Cyt b5) with increased levels of phase II enzymes (GR, GST and GSH) and increased expression of Bax, caspase-3 and caspase-9 with decreased expression of mutated p53 and Bcl-2 in animals treated with DMBA and D-carvone at 20 mg dose. The results of the present study suggest that D-carvone can be used as a chemopreventive agent against skin cancer, as it induces apoptosis in cancer. However, further studies are warranted to check chemopreventive efficacy of D-carvone on cell proliferation, angiogenesis, and metastasis before going to human trial.

RASAGILINE INTERFERES WITH NEURODEGENERATION IN THE PRPH2/RDS MOUSE

Rasagiline (N-propargyl-1(R)-aminoindan) is a second-generation propargylamine with neuroprotective effects. We used the Prph2/rds mouse to assess the effect of rasagiline on photoreceptor cell death and to examine the possible modulation of different pathways of programmed cell death. The animals were orally treated with various doses of rasagiline from Postnatal Day 1 to 56. Methodological approaches consisted of morphometric analyses of the outer nuclear layer thickness and investigation of apoptotic events using TUNEL (TdT-mediated dUTP-biotin nick end labeling) assay, immunohistochemistry, and immunoblot staining. The expression of programmed cell death marker genes involved in photoreceptor degeneration was studied by quantitative real-time polymerase chain reaction. In the Prph2/rds mouse, treatment resulted in a significant dose-dependent neuroprotection at Postnatal Day 56 and a delay in the induction of apoptotic events at Postnatal Day 14. Programmed cell death marker gene expression showed that several mechanisms were involved in photoreceptor degeneration. Furthermore, rasagiline did not only target apoptosis but also other pathways such as autophagy and inflammation. This study showed for the first time significant neuroprotective effects of rasagiline in the retina of Prph2/rds mice through caspase-dependent pathways. However, the activation of caspase-independent programmed cell death pathways that are not affected by rasagiline eventually led to retinal degeneration, but in a delayed manner.

Phenethyl Isothiocyanate (PEITC) Inhibits the Growth of Human Oral Squamous Carcinoma HSC-3 Cells throughG0/G1Phase Arrest and Mitochondria-Mediated Apoptotic Cell Death

Phenethyl isothiocyanate (PEITC), an effective anticancer and chemopreventive agent, has been reported to inhibit cancer cell growth through cell-cycle arrest and induction of apoptotic events in various human cancer cells models. However, whether PEITC inhibits human oral squamous cell carcinoma HSC-3 cell growth and its underlying mechanisms is still not well elucidated. In the present study, we evaluated the inhibitory effects of PEITC in HSC-3 cells and examined PEITC-modulated cell-cycle arrest and apoptosis. The contrast-phase and flow cytometric assays were used for examining cell morphological changes and viability, respectively. The changes of cell-cycle and apoptosis-associated protein levels were determined utilizing Western blotting in HSC-3 cells after exposure to PEITC. Our results indicated that PEITC effectively inhibited the HSC-3 cells' growth and caused apoptosis. PEITC induced G 0/G 1 phase arrest through the effects of associated protein such as p53, p21, p17, CDK2 and cyclin E, and it triggered apoptosis through promotion of Bax and Bid expression and reduction of Bcl-2, leading to decrease the levels of mitochondrial membrane potential (ΔΨm), and followed the releases of cytochrome c, AIF and Endo G then for causing apoptosis in HSC-3 cells. These results suggest that PEITC could be an antitumor compound for oral cancer therapy.

Curcumin decreases cholangiocarcinogenesis in hamsters by suppressing inflammation‐mediated molecular events related to multistep carcinogenesis

Cholangiocarcinoma (CCA) is a highly metastatic tumor linked to liver fluke infection and consumption of nitrosamine-contaminated foods and is a major health problem especially in South-Eastern Asia. In search for a suitable chemopreventive agents, we investigated the effect of curcumin, a traditional anti-inflammatory agent derived from turmeric (Curcuma longa), on CCA development in an animal model by infection with the liver fluke Opisthorchis viverrini and administration of N-nitrosodimethylamine and fed with curcumin-supplemented diet. The effect of curcumin-supplemented diet on histopathological changes and survival were assessed in relation to NF-κB activation, and the expression of NF-κB-related gene products involved in inflammation, DNA damage, apoptosis, cell proliferation, angiogenesis and metastasis. Our results showed that dietary administration of this nutraceutical significantly reduced the incidence of CCA and increased the survival of animals. This correlated with the suppression of the activation of transcription factors including NF-κB, AP-1 and STAT-3, and reduction in the expression of proinflammatory proteins such as COX-2 and iNOS. The formation of iNOS-dependent DNA lesions (8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine) was inhibited. Curcumin suppressed the expression of proteins related to cell survival (bcl-2 and bcl-xL), proliferation (cyclin D1 and c-myc), tumor invasion (MMP-9 and ICAM-1) and angiogenesis (VEGF), and microvessel density. Induction of apoptotic events as indicated by caspase activation and PARP cleavage was also noted. Our results suggest that curcumin exhibits an anticarcinogenic potential via suppression of various events involved in multiple steps of carcinogenesis, which is accounted for by its ability to suppress proinflammatory pathways.

Induction of apoptosis in glioma cells requires cell-to-cell contact with human umbilical cord blood stem cells

We have previously demonstrated the multipotent nature of human umbilical cord blood stem cells (hUCB). In this study, we have attempted to show the use of hUCB in glioma therapy. We used hUCB enriched in CD44 and CD133 cells for our studies and observed that glioma cells co-cultured with hUCB undergo apoptosis. To prove the role of cell-to-cell contact in the induction of apoptotic events, we used a modified 0.22 microm Boyden's chamber where the upper surface was used to culture glioma cells (SNB19 or U87) or xenografts (4910 or 5310) and the lower surface to culture hUCB. TUNEL assay was carried out to determine the degree of apoptotic induction and we observed that glioma or xenograft cells co-cultured with hUCB had a higher number of TUNEL-positive characteristics (63+/-6%) compared to the controls. Further, we co-cultured glioma cells labeled with lipophilic green fluorescent dye and hUCB labeled with lipophilic red fluorescent dye. FACS analysis of cells collected from the upper and lower surfaces revealed that glioma cells had taken up red fluorescent dye from the stem cells (70+/-3%) when compared to glioma cells co-cultured with fibroblast cells (15+/-4%). The apoptotic events in the glioma and xenograft cells co-cultured with hUCB were also confirmed by Western blot analysis for the cleavage of PARP and activation of caspase 8. In addition, elevated levels of CHK-2 levels and downregulation of MAP2K1 were observed in glioma cells co-cultured with hUCB indicating the DNA damage and decrease in cell survival. Nude mice, intracranially implanted with luciferase-expressing U87 cells followed by implantation of hUCB or human fibroblast cells showed retardation of intracranial tumors in hUCB-implanted mice. Taken together, these results demonstrate that hUCB have therapeutic potential with possible clinical implications.

Oral immunoglobulin induces mononuclear cell apoptosis in patients suffering from idiopathic chronic pain syndrome: Results from a pilot study

The objective of this clinical pilot study was to examine the induction of apoptosis in mononuclear cells on treatment of patients with chronic pain syndrome with oral immunoglobulin produced from bovine colostrum (BCC). The 4 patients suffering from chronic idiopathic pain (idiopathic facial pain, CRPS or fibromyalgia) who were enrolled in the study had previously successfully been treated with BCC (varying individual doses). Mononuclear cells from peripheral blood were analyzed for representative cytokines in the serum as well as by TUNEL-assay to detect apoptotic cellular events 14 days after the last treatment with BCC and 14 days after restarting the treatment protocol with BCC. The clinical response (pain and quality of life parameters using a visual analogue scale (VAS)) were determined regularly in each patient. The findings showed a disturbed apoptosis homeostasis in 3 of the 4 patients. These results were accompanied by a relief of the pain symptoms. The 4th patient was found not to need any further analgetic treatment since she demonstrated only nonsignificant changes in her laboratory screening and immunological parameters and by the end of the study she was also completely free of pain (long-term treatment with BCC). In spite of the low patient number, the results were obtained with a sufficiently high degree of control because of the study design. The agreement of the clinical data with our laboratory measurements suggests that the induction of apoptotic events in mononuclear cells is the result of the dominant immunological effects of BCC treatment.