cytochrome-c-t and Kidney-Neoplasms

Renal cell carcinoma (RCC) is well known that it cannot be treated with traditional chemotherapy or radiotherapy. 16-Hydroxycleroda-3,13-dien-15,16-olide (CD), isolated from Polyalthia longifolia Benth. & Hook. f. var. pendula had been reported to display significant efficacy against cancer cell lines.. To determine the anti-tumour activities of CD in two clear cell type RCC (ccRCC) cell lines (A-498 and 786-O). In addition, the underlying mechanisms were also examined.. The cell viabilities of CD-treated ccRCC cells were examined by MTT assay. The apoptotic features were confirmed by acridine orange and ethidium bromide staining. 2',7'-dichlorofluorescin diacetate was used to check reactive oxygen species (ROS) involvement. Mitochondria membrane potential (MMP) were determined by using fluorescent dyes, rhodamine 123 and 5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolylcarbocyanine iodide (JC-1). Proapoptotic, anti-apoptotic proteins and intracellular signaling molecules involved in CD-induced apoptosis were examined by Western blot analysis.. CD inhibited both 786-O and A-498 cell proliferation and induced a series apoptotic characteristics expressions, ROS accumulation, caspase-3 activation as well as poly-(ADP-ribose) polymerase cleavage in both ccRCC cells. Additionally, CD caused MMP reduction and cytochrome c release from mitochondria as well as inhibition of anti-apoptotic proteins, including B cell lymphoma 2 and heat shock protein 70. Mechanically, we address that CD suppressed cell proliferation and induced apoptosis via induction of FOXO3a as well as decreased phosphorylation of Akt, mTOR, MEK/ERK and their downstream molecules, cMyc and hypoxia inducible factor 2α expression in a concentration- and time-dependent trend.. CD caused cell death through ROS overproduction and induction of mitochondria-dependent apoptotic pathway in ccRCC cells that accompanied with multiple oncogenic signals inactivation.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Diterpenes; Humans; Kidney Neoplasms; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; TOR Serine-Threonine Kinases

Nitrogen permease regulator like-2 (NPRL2) has been proved to be a useful suppressor gene in treating many cancers containing renal cancer based on experiments. Transgenic technology which transfect exogenous NPRL2 gene into cancer cell was used in these experiments. However, this technology has defects, such as gene mutation and loss. Cytoplasmic transduction peptide (CTP) can be used to avoid these defects because it can directly mediate proteins to penetrate cell membrane and specifically locate in cytoplasm. In this article, CTP was used to directly mediate NPRL2 protein into the renal cancer cell line 786-O, then cell proliferation was detected by the CCK-8 method, cell cycle and apoptosis were detected by flow cytometry, cell invasion and migration ability were detected by the Transwell assay. Bcl-xl, Cyt-c and caspase-3 were detected by real-time fluorescent quantitative PCR and Western blot for the analysis of the related mechanism. The result showed that CTP successfully mediated NPRL2 protein into renal cancer cells and the growth of cells was significantly inhibited. The mechanism may be NPRL2 down-regulating the expression of Bcl-xl which can up-regulate Cyt-c and further activate caspase-3, and then a cascade reaction is caused for cell apoptosis on the classic mitochondrial apoptosis pathway.

Topics: bcl-X Protein; Carcinoma, Renal Cell; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Recombinant Fusion Proteins; Tumor Suppressor Proteins

Epigenetic modifications play a critical role in the regulation of all DNA-based processes, such as transcription, repair, and replication. Inappropriate histone modifications can result in dysregulation of cell growth, leading to neoplastic transformation and cell death. Renal tumors have been shown to have a higher global methylation percentage and reduced histone acetylation. Preclinical models have revealed that histone gene modifiers and epigenetic alterations play important roles in renal cell carcinoma (RCC) tumorigenesis. Recently, a novel HDAC inhibitor, N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA), has been introduced as an example of a new class of anti-cancer agents. The anti-cancer activity of HNHA and the underlying mechanisms of action remain to be clarified.. The MTS assay using a panel of RCC cells was used to evaluate the anti-proliferative effects of HNHA. The established HDAC inhibitors, SAHA and TSA, were used for comparison. Western blotting analysis was performed to investigate the acetylation of histone H3 and the expression of apoptotic markers in vitro and in vivo. Subcellular fractionation was performed to evaluate expression of Bax and cytochrome c in the cytosol and mitochondria, and also translocation of cytochrome c from the cytoplasm to the nucleus. A confocal microscopic evaluation was performed to confirm inhibition of cell proliferation, induction of apoptosis, and the nuclear translocation of cytochrome c in RCC cells.. In this study, we investigated the apoptosis-inducing activity of HNHA in cultured kidney cancer cells. Apoptosis in the HNHA-treated group was induced significantly, with marked caspase activation and Bcl-2 suppression in RCC cells in vitro and in vivo. HNHA treatment caused cytochrome c release from mitochondria, which was mediated by increased Bax expression and caspase activation. HNHA also induced nuclear translocation of cytochrome c, suggesting that HNHA can induce caspase-independent nuclear apoptosis in RCC cells. An in vivo study showed that HNHA had greater anti-tumor and pro-apoptotic effects on RCC xenografts than the established HDAC inhibitors.. HNHA has more potent anti-tumor activity than established HDAC inhibitors. Its activities are mediated by caspase-dependent and cytochrome-c-mediated apoptosis in RCC cells. These results suggest that HNHA may offer a new therapeutic approach to RCC.

Topics: Acetylation; Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Carcinoma, Renal Cell; Caspases; Cell Fractionation; Cytochromes c; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; I-kappa B Proteins; In Situ Nick-End Labeling; Kidney Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Naphthalenes; Neoplasm Transplantation; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

6-Shogaol, a potent bioactive compound in ginger (Zingiber officinale Roscoe), has been reported for anti-inflammatory and anti-cancer activity. In this study, we investigated the effect of 6-shogaol to enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. The combined treatment with 6-shogaol and TRAIL markedly induces apoptosis in various cancer cells (renal carcinoma Caki cells, breast carcinoma MDA-MB-231 cells and glioma U118MG cells), but not in normal mesangial cells and normal mouse kidney cells. 6-Shogaol reduced the mitochondrial membrane potential (MMP) and released cytochrome c from mitochondria to cytosol via Bax activation. Furthermore, we found that 6-shogaol induced down-regulation of c-FLIP(L) expression at the post-translational levels and the overexpression of c-FLIP(L) markedly inhibited 6-shogaol plus TRAIL-induced apoptosis. Moreover, 6-shogaol increased reactive oxygen species (ROS) production in Caki cells. Pretreatment with ROS scavengers attenuated 6-shogaol plus TRAIL-induced apoptosis through inhibition of MMP reduction and down-regulation of c-FLIP(L) expression. In addition, 6-gingerol, another phenolic alkanone isolated from ginger, did not enhance TRAIL-induced apoptosis and down-regulate c-FLIP(L) expression. Taken together, our results demonstrated that 6-shogaol enhances TRAIL-mediated apoptosis in renal carcinoma Caki cells via ROS-mediated cytochrome c release and down-regulation of c-FLIP(L) expression.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Renal Cell; CASP8 and FADD-Like Apoptosis Regulating Protein; Catechols; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Down-Regulation; Drug Screening Assays, Antitumor; Humans; Kidney Neoplasms; Mice; Reactive Oxygen Species; Structure-Activity Relationship; TNF-Related Apoptosis-Inducing Ligand

A metal wire-inserted disposable gel-loading tip was examined as an electrospray emitter. Its performance was similar to that of conventional electrospray ionization (ESI) with a relatively low flow rate (~100 nL/min) and without the need for solvent pumps. It was also used as an emitter for solid probe-assisted ESI (SPA-ESI) (e.g., biofluid was sampled from the biological tissue by a needle and was inserted into the solvent-preloaded gel-loading tip). Selective detection of lipids and proteins, such α and β chains of hemoglobin could be accomplished by choosing appropriate solvents. A suitable protocol for cancer diagnosis was established by this method. A good figure of merit of this method is its applicability to biological tissue diagnostics with high cost efficiency and on a disposable basis.

Topics: beta-Globins; Carcinoma, Renal Cell; Cytochromes c; Equipment Design; Hemoglobins; Humans; Insulin; Kidney; Kidney Neoplasms; Needles; Peptide Fragments; Principal Component Analysis; Spectrometry, Mass, Electrospray Ionization; Ubiquitin

The aim of this study was to determine the molecular mechanisms of physalin F, an effective purified extract of Physalis angulata L. (Solanacae), in renal carcinoma A498 cells.. Physalin F was observed to significantly induce cytotoxicity of three human renal carcinoma A498, ACHN, and UO-31 cells in a concentration-dependent manner; this was especially potent in A498 cells. The physalin F-induced cell apoptosis of A498 cells was characterized by MTT assay, nuclear DNA fragmentation and chromatin condensation. Using flow cytometry analysis, physalin F induced A498 cell apoptosis as demonstrated by the accumulation of the sub-G1 phase in a concentration- and time-dependent manner. Moreover, physalin F-mediated accumulation of reactive oxygen species (ROS) caused Bcl-2 family proteins, Bcl-2, and Bcl-xL degradation, which led to disruption of mitochondrial membrane potential and release of cytochrome c from the mitochondria into the cytosol. These effects were associated with induction of caspase-3 and caspase-9 activity, which led to poly(ADP-ribose) polymerase cleavage. However, the antioxidant N-acetyl-(L)-cysteine (NAC) and glutathione (GSH) resulted in the inhibition of these events and reversed physalin F-induced cell apoptosis. In addition, physalin F suppressed NF-κB activity and nuclear translocation of p65 and p50, which was reversed by NAC and GSH.. Physalin F induced cell apoptosis through the ROS-mediated mitochondrial pathway and suppressed NF-κB activation in human renal cancer A498 cells. Thus, physalin F appears to be a promising anti-cancer agent worthy of further clinical development.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Cell Cycle; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; Kidney Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; NF-kappa B; Phytotherapy; Reactive Oxygen Species; Secosteroids

Renal cell carcinoma (RCC) is the most frequent type of renal-originated malignancy. Although nephrectomy is successfully used to save the lives of patients with localized RCC, treatment of advanced and other refractory RCCs is poor and still inadequate. Here, we show that triptolide, a small molecule and a well-known anti-inflammatory and anti-immunity agent used in the clinic, is capable of inducing cell apoptosis via the mitochondrial pathway in the 786-0 RCC cell line. This induction occurred in concert with reduced expression of genes related to the stabilization of mitochondria such as Bcl-2 and Bcl-XL. Cell cycle analysis showed that exposure to triptolide decreased the proportion of cells in the G0/G1 and G2/M phases, and increased the proportion of cells in the S phase. Cell accumulation in the S phase can be attributed to reduced expression of cell cycle checkpoint regulators such as cyclin A, cyclin B, CDK1, CDK2 and retinoblastoma proteins (Rb). These results raise the possibility that triptolide-induced apoptosis is mediated by cell cycle arrest. Similarly, in another human RCC cell line, OS-RC-2, triptolide-induced apoptosis and cell accumulation in S phase were also observed. Therefore, triptolide emerges as a stimulator of apoptosis by influencing coordinate regulation of proliferation and apoptosis, and may be applicable to the treatment of human renal cell carcinoma.

Topics: Antineoplastic Agents, Alkylating; Apoptosis; Carcinoma, Renal Cell; Caspases; Cell Cycle; Cytochromes c; Diterpenes; Epoxy Compounds; Humans; Kidney Neoplasms; Mitochondria; Phenanthrenes; Tumor Cells, Cultured

Cafestol, one of the major compounds in coffee beans, has been reported for its tumor cell growth inhibitory activity and anti-carcinogenic activity, although the mechanism of action is poorly understood. In the present study, we investigated the effect of cafestol on the apoptotic pathway in human renal Caki cells and other cancer cell lines. Cafestol treatment inhibited Caki cells viability a dose-dependent manner by inducing apoptosis, as evidenced by DNA fragmentation and the accumulation of sub-G1 phase. Cafestol-induced apoptosis is associated with the reduction of mitochondrial membrane potential (MMP), activation of caspase 3, cytochrome c release, and down-regulation of anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1 and cFLIP). Cafestol-induced apoptosis was blocked by pretreatment with broad caspase inhibitor z-VAD-fmk, showing its dependence on caspases. Ectopic expression of Bcl-2 or Mcl-1 in Caki cells attenuates cafestol-induced apoptosis. In addition, we have also shown that cafestol inhibits phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, and PI3K inhibitor LY29004 significantly increases cafestol-induced apoptosis in Caki cells. Taken together, our results show the activity of cafestol to modulate multiple components in apoptotic response of human renal Caki cells and a potential as a therapeutic agent for preventing cancers such as renal carcinoma.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Renal Cell; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Chromones; Cytochromes c; Diterpenes; DNA Fragmentation; Down-Regulation; G1 Phase; Humans; Kidney Neoplasms; Membrane Potential, Mitochondrial; Morpholines; Myeloid Cell Leukemia Sequence 1 Protein; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

We previously elucidated an important role for gangliosides in renal cell carcinoma-mediated T lymphocyte apoptosis, although the mechanism by which they mediated lymphocyte death remained unclear. Here, we show that when added in purified form, GD3 is internalized by activated T cells, initiating a series of proapoptotic events, including the induction of reactive oxygen species (ROS), an enhancement of p53 and Bax accumulation, an increase in mitochondrial permeability, cytochrome c release, and the activation of caspase-9. GD3-induced apoptosis of activated T cells was dose dependent and inhibitable by pretreating the lymphocytes with N-acetylcysteine, cyclosporin A, or bongkrekic acid, emphasizing the essential role of ROS and mitochondrial permeability to the process. Ganglioside-induced T-cell killing was associated with the caspase-dependent degradation of nuclear factor-kappaB-inducible, antiapoptotic proteins, including RelA; this suggests that their loss is initiated only after the cascade is activated and that their disappearance amplifies but not triggers GD3 susceptibility. Resting T cells did not internalize appreciable levels of GD3 and did not undergo any of the proapoptotic changes that characterize activated T lymphocytes exposed to the ganglioside. RelA overexpression endows Jurkat cells with resistance to GD3-mediated apoptosis, verifying the role of the intact transcription factor in mediating protection from the ganglioside.

Topics: Acetylcysteine; Antioxidants; Apoptosis; Carcinoma, Renal Cell; Caspase 8; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Cell Membrane Permeability; Cytochromes c; Gangliosides; Glioblastoma; Humans; Jurkat Cells; Kidney Neoplasms; Lymphocyte Activation; Mitochondrial Membranes; Reactive Oxygen Species; T-Lymphocytes

We have reported that connexin (Cx) 32 gene, a member of gap junction protein family, acts as a tumor suppressor gene in human renal cell carcinoma (RCC). Of solid tumors, RCC is one of the most chemoresistant cancers, and there is no effective cancer chemotherapy against RCC at present. In this study, we examined if the combination of Cx32-dependent tumor-suppressive effect and vinblastine (VBL), a chemotherapeutic agent which has been utilized for clinical RCC treatment, could be effective in enhancing the sensitivity of RCC to VBL treatment. Cx32 expression in a human metastatic RCC cell (Caki-1 cell) significantly enhanced in vitro and in vivo VBL-induced cytotoxicity on the cell. Cx32 expression in the RCC cells potentiated VBL-induced apoptosis compared to the Cx32-negative RCC cells in vitro as well as in vivo. The enhancing apoptosis in the RCC cells by Cx32 mainly depended on the decrease of P-glycoprotein (P-gp), a multidrug resistance gene-1 (MDR-1) product responsible for reduction of VBL accumulation into the cells. We also observed that silencing of Cx32 by short interfering RNA (siRNA) treatment elevated the level of P-gp in Caki-1 cells and that inhibition of P-gp function enhanced VBL-induced apoptosis in the RCC cells. These results suggest that Cx32 is effective to enhance VBL-induced cytotoxicity in Caki-1 cells via the reduction of P-gp. Overall, it seems that the combination of Cx32-dependent tumor-suppressive effect and VBL is promising as a new cancer therapy against RCC.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Renal Cell; Cell Cycle; Cell Proliferation; Combined Modality Therapy; Connexins; Cytochromes c; Drug Resistance, Multiple; Gap Junction beta-1 Protein; Humans; Kidney Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Tumor Cells, Cultured; Vinblastine

The kidney is subjected to DNA oxidative damage from reactive oxygen species generated by free radicals and toxic metabolites, leading to formation of DNA base lesions. One such DNA lesion is 8-oxoguanine, which, if not sufficiently removed, is potentially mutagenic because it can cause G:C to T:A transversion in subsequent DNA replication. The human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene on chromosome 3, a region (3p25-26) that shows frequent loss of heterozygosity in clear cell renal cell carcinoma (CC-RCC), encodes for a DNA repair enzyme capable of excision repair of 8-oxoguanine. Of the known isoforms of the hOGG1 enzyme (types Ia, Ib, Ic, Id, and II), only 1, Ia, is found in the nucleus, whereas the rest show a mitochondrial distribution. We investigated, by an immunohistochemical staining method, the expression of hOGG1 protein in 40 cases of CC-RCC, using archival formalin-fixed tissue. To localize the hOGG1 enzyme in normal and tumor tissue, immuno-staining against cytochrome c, a specific mitochondrial enzyme, was also performed. The results showed marked reduction in hOGG1 expression in the majority of tumors, with complete loss of staining seen in 26 (65%) and moderate and weak positive staining present in 9 (22.5%) and 5 (12.5%) of the cases, respectively. Strong hOGG1 protein expression was present in normal tubular epithelium, located in the mitochondria. The results correlated with the expression patterns of cytochrome c. The findings indicate that loss of hOGG1 expression may have a role in development or progression of CC-RCC.

Topics: Adenocarcinoma, Clear Cell; Cytochromes c; DNA Glycosylases; DNA Repair; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Isoenzymes; Kidney Neoplasms; Mitochondria; Mitochondrial Proteins