BAG-1 promotes apoptosis induced by N-(4-hydroxyphenyl)retinamide in human cervical carcinoma cells

N-(4-hydroxyphenyl)retinamide (4-HPR) is a synthetic apoptosis-inducing retinoid with cancer chemopreventive properties and lower toxicity than all-trans retinoic acid. BAG-1 is an antiapoptotic gene that is overexpressed in cervical and other cancers. In this study, we examined whether BAG-1 can inhibit 4-HPR-induced apoptosis in the C33A cervical carcinoma cell line. Surprisingly, although it inhibited apoptosis induced by five different apoptotic stimuli, overexpression of BAG-1 enhanced apoptosis induced by 4-HPR, producing a 2.5-fold lower IC(50) of 4-HPR. The effects of BAG-1 on 4-HPR-induced apoptosis were mediated by enhancing the caspase-3 activation pathway. Deletion mutation experiments showed that the central ubiquitin homology domain of BAG-1 protein was necessary for its promotion of 4-HPR-induced apoptosis, whereas its C-terminal Hsp70/Hsc70-interacting domain was required for its inhibition of staurosporine-induced apoptosis. These in vitro results suggest that the effectiveness of 4-HPR against the development of malignancy may be due to the overexpression of BAG-1 in cancer cells.     

BAG-1 Promotes Apoptosis Induced by N-(4-hydroxyphenyl)retinamide in Human Cervical Carcinoma Cells

N-(4-hydroxyphenyl)retinamide (4-HPR) is a synthetic apoptosis-inducing retinoid with cancer chemopreventive properties and lower toxicity than all-trans retinoic acid. BAG-1 is an antiapoptotic gene that is overexpressed in cervical and other cancers. In this study, we examined whether BAG-1 can inhibit 4-HPR-induced apoptosis in the C33A cervical carcinoma cell line. Surprisingly, although it inhibited apoptosis induced by five different apoptotic stimuli, overexpression of BAG-1 enhanced apoptosis induced by 4-HPR, producing a 2.5-fold lower IC₅₀ of 4-HPR. The effects of BAG-1 on 4-HPR-induced apoptosis were mediated by enhancing the caspase-3 activation pathway. Deletion mutation experiments showed that the central ubiquitin homology domain of BAG-1 protein was necessary for its promotion of 4-HPR-induced apoptosis, whereas its C-terminal Hsp70/Hsc70-interacting domain was required for its inhibition of staurosporine-induced apoptosis. These in vitro results suggest that the effectiveness of 4-HPR against the development of malignancy may be due to the overexpression of BAG-1 in cancer cells.     

4-氨基苯酚维甲酰胺对肝癌和恶性黑色素瘤细胞的抑制作用

In this paper, a significantly effect of N-(4-hydrophenyl) retinamide (4-HPR), a derivative of retinoic acid, was observed on inhibition of migration, invasion, cell growth, and induction of apoptosis in hepatoma cells and B16 melanoma cells. The number of migratory hepatoma cells reduced significantly from the control 201 +/- 27.2 to 58 +/- 5.03 after 6-hour incubation with 4-HPR (p < 0.01, n = 4). The number of migratory B16 melanoma cells reduced from the control 302 +/- 30.1 to 254 +/- 25.04 (p < 0.05, n = 4). The invasive ability of these cells was also suppressed by 4-HPR treatment. Cells that penetrated the artificial membrane matrigel decreased from 27 +/- 13.1 to 11.2 +/- 3.3 in hepatoma cells, from 67.5 +/- 10.1 to 24.3 +/- 3.2 in B16 melanoma cells (p < 0.05, n = 3). Furthermore, cell growth was significantly inhibited especially in B16 melanoma cells and 37.11 +/- 0.94% cells were induced to apoptosis after 48-hour induction by 4-HPR, which was significantly higher than those by retinoic acid treatment (p < 0.05). Although the mechanism of 4-HPR effects was not very clear, over expression of CST, which was inhibited by 4-HPR in our previous study, could diminish the apoptosis--inducing effect by 4-HPR. We believe that 4-HPR has a strong inhibitory effect on melanoma and hepatocarcinoma cells and might become a potent therapeutic agent.     

Ceramide signaling in fenretinide-induced endothelial cell apoptosis

Stress stimuli can mediate apoptosis by generation of the lipid second messenger, ceramide. Herein we investigate the molecular mechanism of ceramide signaling in endothelial apoptosis induced by fenretinide (N-(4-hydroxyphenyl)retinamide (4-HPR)). 4-HPR, a synthetic derivative of retinoic acid that induces ceramide in tumor cell lines, has been shown to have antiangiogenic effects, but the molecular mechanism of these is largely unknown. We report that 4-HPR was cytotoxic to endothelial cells (50% cytotoxicity at 2.4 microm, 90% at 5.36 microm) and induced a caspase-dependent endothelial apoptosis. 4-HPR (5 microm) increased ceramide levels in endothelial cells 5.3-fold, and the increase in ceramide was required to achieve the apoptotic effect of 4-HPR. The 4-HPR-induced increase in ceramide was suppressed by inhibitors of ceramide synthesis, fumonisin B(1), myriocin, and l-cycloserine, and 4-HPR transiently activated serine palmitoyltransferase, demonstrating that 4-HPR induced de novo ceramide synthesis. Sphingomyelin levels were not altered by 4-HPR, and desipramine had no effect on ceramide level, suggesting that sphingomyelinase did not contribute to the 4-HPR-induced ceramide increase. Finally, the pancaspase inhibitor, t-butyloxycarbonyl-aspartyl[O-methyl]-fluoromethyl ketone, suppressed 4-HPR-mediated apoptosis but not ceramide accumulation, suggesting that ceramide is upstream of caspases. Our results provide the first evidence that increased ceramide biosynthesis is required for 4-HPR-induced endothelial apoptosis and present a molecular mechanism for its antiangiogenic effects.     

N-(4-hydroxyphenyl)retinamide (4-HPR) decreases neoplastic properties of human prostate cells: an agent for prevention

The development of prostate cancer through a multistep process of carcinogenesis may have a long latent period of 20-30 years. It is possible that progression to a malignant state could be blocked or reversed during this time. This study focuses on the ability of the synthetic retinoid, N-(4-hydroxyphenyl)-retinamide (4-HPR), to reverse changes associated with malignant transformation and tumor progression, towards a normal phenotype. To examine the responsiveness of cells at different steps of prostate carcinogenesis, three immortalized, but non-tumorigenic (RWPE-1, WPE1-7 and WPE1-10), and one human prostate carcinoma cell line (DU-145), were used. The effects of 4-HPR on cell proliferation, expression of intermediate filament proteins cytokeratin 18 and vimentin, and tumor suppressor proteins p53 and pRb were examined by immunostaining and compared. Results show that 4-HPR caused inhibition of growth in all cell lines in a dose-dependent manner. 4-HPR induced an increase in staining for cytokeratin 18, a marker of differentiation for prostate epithelial cells. While all cell lines showed strong immunostaining for vimentin, treatment with 4-HPR for 8 days caused a marked decrease in staining for vimentin in all cell lines. In an in vitro assay, 4-HPR also caused inhibition of invasion by DU-145 cells in a dose-dependent manner. Furthermore, 4-HPR treatment was effective in significantly decreasing the abnormal nuclear staining for the tumor suppressor proteins p53 and pRb. Because 4-HPR decreased invasion-associated vimentin expression, inhibited invasion, and normalized p53 and pRb immunostaining, we propose that 4-HPR may be an effective agent for secondary and tertiary prevention, i.e. promotion and progression stages, respectively, of prostate cancer.     

Comparison of the mechanism of induction of apoptosis in ovarian carcinoma cells by the conformationally restricted synthetic retinoids CD437 and 4-HPR

All-trans-retinoic acid (ATRA) has been shown to inhibit the growth of a number of ovarian tumor cell lines while others have been found to be resistant to retinoid suppression of growth. Interestingly, two synthetic retinoids, CD437 and 4-HPR, inhibit the growth of both ATRA-sensitive (CA-OV-3) and ATRA-resistant (SK-OV-3) ovarian tumor cells. However, in contrast to ATRA, both induce apoptosis. Our goal was to elucidate the mechanism by which these two synthetic retinoids induce apoptosis in ovarian tumor cells. Since it has been documented that apoptosis induction is often mediated by the activation of a cascade of proteases known as caspases, we initially studied the role of caspases in induction of apoptosis by CD437 and 4-HPR. We found that both retinoids induced caspase-3 and caspase-9 enzyme activity. Furthermore, using caspase specific inhibitors we determined that caspase-3 and caspase-9 activity was essential for the induction of apoptosis by these synthetic retinoids since these inhibitors completely blocked CD437 and 4-HPR induced apoptosis. Interestingly, we found that treatment with bongkriekic acid (BA), a mitochondrial membrane depolarization inhibitor, blocked apoptosis, caspase-9 activation and caspase-3 activation induced by both retinoids. Finally, we were able to determine that CD437 treatment induced the translocation of TR3, a nuclear orphan receptor, whereas, 4-HPR did not. Our results suggest that CD437 and 4-HPR initially activate separate pathways to induce mitochondrial depolarization but both utilize mitochondrial depolarization, caspase-9 activation, and caspase-3 activation in the later stages of apoptosis induction.     

Synthetic retinoids as inducers of apoptosis in ovarian carcinoma cell lines

Apoptosis is also known as programmed cell death. Apoptosis plays an essential role in maintaining normal tissue and cell physiology in multicellular organisms. Clearance of aberrant or pre-cancerous cells occurs through the induction of apoptosis. It has been reported that many tumors and tumor cell lines have dysfunctional apoptosis signaling, causing these tumors to escape immune monitoring and internal cellular control mechanisms. One potential cause of this dysfunctional apoptosis is the tumor suppressor p53, an important regulator of growth arrest and apoptosis that is mutated in over 50% of all cancers. Retinoids have great potential in the areas of cancer therapy and chemoprevention. While some tumor cells are sensitive to the growth inhibitory effects of natural retinoids such as all-trans-retinoic acid (ATRA), many ovarian tumor cells are not. 6-[3-(1-Admantyl)]-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) and fenretinide N-[4-hydroxyphenyl] retinamide (4-HPR) are conformationally restricted synthetic retinoids that induce growth arrest and apoptosis in both ATRA-sensitive and ATRA-resistant ovarian tumor cell lines. Recently, we have identified the molecular pathways of apoptosis induced by treatment of ovarian carcinoma cells with mutated p53 by CD437 and 4-HPR.     

Human bcl-2 gene attenuates the ability of rabbit lens epithelial cells against H2O2-induced apoptosis through down-regulation of the alpha B-crystallin gene

It is well established that the proto-oncogene, bcl-2, can prevent apoptosis induced by a variety of factors. Regarding the mechanism by which BCL-2 prevents cell death, one theory suggests that it acts by protecting cells from oxidative stress. In the lens system, oxidative stress-induced apoptosis is implicated in cataractogenesis. To explore the possibility of anti-apoptotic gene therapy development for cataract prevention and also to further test the anti-oxidative stress theory of BCL-2 action, we have introduced the human bcl-2 gene into an immortalized rabbit lens epithelial cell line, N/N1003A. The stable expression clones of both vector- and bcl-2-transfected cells have been established. Treatment of the two cell lines with H(2)O(2) revealed that bcl-2-transfected cells were less capable of detoxifying H(2)O(2) than the control cells. Moreover, bcl-2-transfected cells are more susceptible to H(2)O(2)-induced apoptosis. To explore why bcl-2-transfected cells have reduced resistance to H(2)O(2)-induced apoptosis, we examined the expression patterns of several relevant genes and found that expression of the alphaB-crystallin gene was distinctly down-regulated in bcl-2-transfected cells compared with that in vector-transfected cells. This down-regulation was specific because a substantial inhibition of BCL-2 expression through antisense bcl-2 RNA significantly restored the level of alphaB-crystallin and, moreover, enhanced the ability of the bcl-2-transfected cells against H(2)O(2)-induced apoptosis. Introduction of a mouse alphaB-crystallin gene into bcl-2-transfected cells also counteracted the BCL-2 effects. Down-regulation of alphaB-crystallin gene was largely derived from changed lens epithelial cell-derived growth factor activity. Besides, alphaB-crystallin prevents apoptosis through interaction with procaspase-3 and partially processed procaspase-3 to prevent caspase-3 activation. Together, our results reveal that BCL-2 can regulate gene expression in rabbit lens epithelial cells. Through down-regulation of the alphaB-crystallin gene, BCL-2 attenuates the ability of rabbit lens epithelial cells against H(2)O(2)-induced apoptosis.     

Alkaline Ceramidase 2 (ACER2) and Its Product Dihydrosphingosine Mediate the Cytotoxicity of N-(4-Hydroxyphenyl)retinamide in Tumor Cells

Increased generation of dihydrosphingosine (DHS), a bioactive sphingolipid, has been implicated in the cytotoxicity of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in tumor cells. However, how 4-HPR increases DHS remains unclear. Here we demonstrate that 4-HPR increases the expression of ACER2, which catalyzes the hydrolysis of dihydroceramides to generate DHS, and that ACER2 up-regulation plays a key role in mediating the 4-HPR-induced generation of DHS as well as the cytotoxicity of 4-HPR in tumor cells. Treatment with 4-HPR induced the accumulation of dihydroceramides (DHCs) in tumor cells by inhibiting dihydroceramide desaturase (DES) activity, which catalyzes the conversion of DHCs to ceramides. Treatment with 4-HPR also increased ACER2 expression through a retinoic acid receptor-independent and caspase-dependent manner. Overexpression of ACER2 augmented the 4-HPR-induced generation of DHS as well as 4-HPR cytotoxicity, and 4-HPR-induced death in tumor cells, whereas knocking down ACER2 had the opposite effects. ACER2 overexpression, along with treatment with GT11, another DES inhibitor, markedly increased cellular DHS, leading to tumor cell death, whereas ACER2 overexpression or GT11 treatment alone failed to do so, suggesting that both ACER2 up-regulation and DES inhibition are necessary and sufficient to mediate 4-HPR-induced DHS accumulation, cytotoxicity, and death in tumor cells. Taken together, these results suggest that up-regulation of the ACER2/DHS pathway mediates the cytotoxicity of 4-HPR in tumor cells and that up-regulating or activating ACER2 may improve the anti-cancer activity of 4-HRR and other DHC-inducing agents.     

重组RA—538及反义c—myc腺病毒对人胃癌,食管癌和bcl—2高表达细胞系的作用

本课题研究ＲＡ５３８、反义c-ymc重组腺病毒对人胃癌（ＳＧＣ７９０１）、食管癌（Ｅ Ｃ１０９、ＥＣ８７１２）、正常人胚肺２ＢＳ（２ＢＳ）及ｂｃｌ－２高表达细胞第的体仙外生物学作用及其分子机制。结果显示Ａｄ－ＲＡ５３８及Ａｄ－ＡＳｃ－ｍｙｃ对ＳＧＣ７９０１细胞体内外均具有明显的生长抑制及凋亡诱导作用,并能抑制其ｃ－ｍｙｃ、ｂｃｌ－２、ｃｙｃｌｉｎＤ１基因的表达及刺激ｂａｘ基因的表达。对ＥＣ１０９、ＥＣ８     

Immunohistochemical analysis for cell proliferation-related protein expression in small cell carcinoma of the esophagus; a comparative study with small cell carcinoma of the lung and squamous cell carcinoma of the esophagus

Small cell carcinoma is a rare neoplasm in the esophagus. To evaluate cell proliferation activity and its underlying mechanisms in this tumor, we examined immunohistochemically 5 cases of small cell carcinoma of the esophagus (SCCE) for expressions of tumor suppressor proteins, oncoproteins and cell proliferation markers including p53, p21WAF1/CIP1, retinoblastoma (Rb) protein, bcl-2, Ki-67 and PCNA, and compared the results with those of 5 cases of small cell carcinoma of the lung (SCCL) and 10 cases of squamous cell carcinoma of the esophagus (SQCE). The prevalence and labeling index of p53-immunoreactivity tended to be higher in SCCE (4/5; 56.6%) and SCCL (4/5; 79.9%) than in SQCE (6/10; 48.8%). Expression of p21WAF1/CIP1 was observed in 2 of 10 cases of SQCE. In contrast, its expression could not be detected in any cases of SCCE and SCCL examined. Expression of Rb protein was observed in 9 out of 10 cases of SQCE, but not in any cases of SCCE and SCCL. SCCE and SCCL showed more frequent and intense immunoreactivity for bcl-2 than SQCE. In expression of cell proliferation markers (Ki-67 and PCNA), no remarkable difference was observed among SCCE, SCCL and SQCE. These results suggest that SCCE and SCCL could share some genetic alternations including mutation of p53, loss of Rb gene and overexpression of bcl-2, and these may be related to the similar biological potentials between the two. Furthermore, SCCE was different from SQCE in expression of Rb protein and bcl-2, and these two types of esophageal carcinoma could arise through different molecular mechanisms.     

Involvement of histone hypoacetylation in Ni<Superscript>2+</Superscript>-induced<Emphasis Type="Italic"> bcl</Emphasis>-<Emphasis Type="Italic">2</Emphasis> down-regulation and human hepatoma cell apoptosis

Although induction of cell apoptosis is known to be involved in the cytotoxicity of Ni(2+), little research has been aimed at the mechanism of Ni(2+)-induced apoptosis. Recent studies showed that Ni(2+) induces histone hypoacetylation in different cell lines. Since histone hypoacetylation plays important roles in the control of cell cycle progress and apoptosis, we hypothesized that histone hypoacetylation may be an unrevealed pathway in Ni(2+)-induced apoptosis. To address this, effects of Ni(2+) on cell apoptosis, bcl- 2 gene expression and histone acetylation were examined in human hepatoma Hep3B cells. We found that Ni(2+) treatment resulted in cell proliferation arrest, the appearance of detached cells, condensed chromatin, apoptotic bodies and specific DNA fragmentation, indicating the occurrence of cell apoptosis. At the same time, Ni(2+) induced a significant decrease in bcl- 2 expression and histone acetylation; the decrease of histone H4 acetylation in nucleosomes associated with the bcl- 2 promoter region was also proven by a chromatin immunoprecipitation assay, indicating the involvement of histone hypoacetylation in Ni(2+)-induced bcl- 2 down-regulation. Further studies showed that increasing histone acetylation by either 100 nM of trichostatin A or over-expressing histone acetyltranferase p300 in Hep3B cells obviously attenuated the bcl- 2 down-regulation and cell apoptosis caused by Ni(2+). Considering the importance of bcl- 2 in determining cell survival and apoptosis, the data presented here suggest that histone hypoacetylation may represent one unrevealed pathway in Ni(2+)-induced cell apoptosis, where bcl- 2 is one of its targets.     

Hydrolysis of 4-HPR to atRA occurs in vivo but is not required for retinamide-induced apoptosis

The retinamide, N-(4-hydroxyphenyl)retinamide (4-HPR), has shown promising anti-tumor activity, but it is unclear whether this compound is hydrolyzed to all-trans retinoic acid (atRA) and if so, whether this plays any role in its chemotherapeutic activity. To address this issue, the ability of 4-hydroxybenzylretinone (4-HBR), a carbon-linked analog of 4-HPR, to support growth in vitamin A-deficient (VAD) animals and to activate an atRA-responsive gene in vivo was compared to 4-HPR and atRA. Further, the non-hydrolyzable 4-HBR analog was used to determine whether the presence of the labile amide linkage in 4-HPR is essential for the induction of apoptosis in cultured MCF-7 breast cancer cells. Studies in VAD rats showed that 4-HPR, like atRA, supports animal growth and induces CYP26B1 mRNA expression in lung whereas 4-HBR does not. Analysis of plasma from 4-HPR- and atRA-treated VAD animals revealed the presence of atRA whereas it was not detected in plasma from animals given 4-HBR. To determine whether hydrolysis to atRA is necessary for apoptosis induced by 4-HPR in MCF-7 breast cancer cells, morphological and biochemical assays for apoptosis were performed. 4-HBR, like 4-HPR, induced apoptosis in MCF-7 cells. Apoptosis was not induced even at high concentrations of atRA, showing that 4-HPR and 4-HBR act in cells via a distinct signaling pathway. These results show that although limited hydrolysis of 4-HPR occurs in vivo, the ability to liberate atRA is not required for these 4-hydroxyphenyl retinoids to induce apoptosis in MCF-7 breast cancer cells. Thus the non-hydrolyzable analog, 4-HBR, may have significant therapeutic advantage over 4-HPR because it does not liberate atRA that can contribute to the adverse side effects of drug administration in vivo.     

Down-regulation of Mcl-1 by inhibition of the PI3-K/Akt pathway is required for cell shrinkage-dependent cell death.

The anti-apoptotic effect of a chloride-bicarbonate exchange blocker has been previously examined in endothelial cells and cardiomyocytes. However, the anti-apoptotic effects of this blocker on epithelial cells and the mechanism of the anti-apoptotic effect remain unknown. We examined the anti-apoptotic effects of a chloride-bicarbonate exchange blocker in a renal epithelial cell line (MDCK cells). Changes in the expression of bcl-2 family proteins, which are known to have anti-apoptotic effects, were also examined. Staurosporine was used to induce apoptotic cell death in the MDCK cells. Staurosporine treatment was sufficient to induce apoptotic cell death, detected by propidium iodide and DNA ladder formation. A chloride-bicarbonate exchange blocker was added 24 h before the staurosporine treatment and during treatment. The chloride-bicarbonate exchange blocker inhibited the staurosporine-induced apoptosis in the MDCK cells in a dose-dependent manner. The expression of bcl-2 family gene products was detected by RT-PCR and Western blotting. No changes in the expression of Bax, Bid and Bik (pro-apoptotic proteins), or Bcl-2 (an anti-apoptotic protein) were detected. However, Mcl-1 expression was reduced by the staurosporine treatment, and this reduction was recovered when the chloride-bicarbonate exchange blocker was added. LY294002, a PI 3-kinase inhibitor, partially inhibited this anti-apoptotic effect. In conclusion, chloride-bicarbonate exchange blockers appear to offer cell-protective effects via Mcl-1 up-regulation.     

Dihydroceramide accumulation and reactive oxygen species are distinct and nonessential events in 4-HPR-mediated leukemia cell death

4-(Hydroxyphenyl)retinamide (4-HPR) is a synthetic retinoid with a strong apoptotic effect towards different cancer cell lines in vitro, and it is currently tested in clinical trials. Increases of reactive oxygen species (ROS) and modulation of endogenous sphingolipid levels are well-described events observed upon 4-HPR treatment, but there is still a lack of understanding of their relationship and their contribution to cell death. LC-MS analysis of sphingolipids revealed that in human leukemia CCRF-CEM and Jurkat cells, 4-HPR induced dihydroceramide but not ceramide accumulation even at sublethal concentrations. Myriocin prevented the 4-HPR-induced dihydroceramide accumulation, but it did not prevent the loss of viability and increase of intracellular ROS production. On the other hand, ascorbic acid, Trolox, and vitamin E reversed 4-HPR effects on cell death but not dihydroceramide accumulation. NDGA, described as a lipoxygenase inhibitor, exerted a significantly higher antioxidant activity than vitamin E and abrogated 4-HPR-mediated ROS. It did not however rescue cellular viability. Taken together, this study demonstrates that early changes observed upon 4-HPR treatment, i.e., sphingolipid modulation and ROS production, are mechanistically independent events. Furthermore, the results indicate that 4-HPR-driven cell death may occur even in the absence of dihydroceramide or ROS accumulation. These observations should be taken into account for an improved design of drug combinations.     

Silencing of RHEB inhibits cell proliferation and promotes apoptosis in colorectal cancer cells via inhibition of the mTOR signaling pathway

Colorectal cancer (CRC) is commonly known as one of the most prominent reasons for cancer-related death in China. Ras homolog enriched in brain (RHEB) and the mammalian target activity of rapamycin (mTOR) signaling pathway were found correlated with CRC, but their specific interaction in CRC was still to be investigated. Therefore, we explored whether RHEB gene silencing affected the cell proliferation, differentiation, and apoptosis by directly targeting the mTOR signaling pathway in cells previously harvested from CRC patients. A microarray analysis was subsequently conducted to investigate the relationship between RHEB and mTOR. Eighty-three adjacent normal tissues and CRC tissues were selected. Immunohistochemistry was carried out to detect the positive expression rates of RHEB and Ki-67 in the CRC tissues. Cells were then transfected with different siRNAs to investigate the potential effects RHEB would have on CRC progression. The expressions of RHEB, 4EBP1, ribosomal protein S6 kinase (p70S6K), proliferating cell nuclear antigen (PCNA), B cell lymphoma 2 (bcl-2), and bcl-2-associated X protein (bax) were determined and then the cell cycle, cell proliferation, and apoptotic rate were also measured. We identified RHEB and mTOR as upregulated genes in CRC. Cells treated with RHEB silencing showed a decreased extent of mTOR, p70S6K, 4EBP1 phosphorylation and expression of RHEB, Ki-67, mTOR, p70S6K, 4EBP1, bcl-2, and PCNA as well as decreased activity of cell proliferation and differentiation; although, the expression of bax was evidently higher. Collectively, our data propose the idea that RHEB gene silencing might repress cell proliferation and differentiation while accelerating apoptosis via inactivating the mTOR signaling pathway.     

Role of vitamins in determining apoptosis and extent of suppression by bcl-2 during hybridoma cell culture

The identification of cell culture media components that may instigate apoptosis in cell lines used for the production of commercial antibodies and recombinant proteins, is crucial to aid the development of improved media for reduced cell death and to understand the role of nutrient components in cell survival and maintenance. Here we determine the impact of depriving all or individual B-group media vitamins either, D-CaPantothenate (DCaP), choline chloride (CC), riboflavin (Rb), i-inositol, nicotinamide (NAM), pyridoxal hydrochloride (PyrHCl), folic acid (FA), or thiamine hydrochloride (ThHCl) on hybridoma cell growth and viability using fluorescence microscopy techniques. Cultivation in media deprived of all these vitamins prevented cell proliferation from reaching maximum capacity while increasing cell death rate, predominantly via apoptosis. Deletion of either DCaP, CC, or Rb showed that these components were most likely responsible for the development of apoptosis. Exclusion of either i-inositol, NAM or PyrHCl failed to inhibit cell growth and viability, while marginal improvements in viability were noted by ThHCl deprivation and more so by FA exclusion. Over-expression of the anti-apoptotic gene bcl-2 suppressed cell death initiated by all or single vitamin (either DCaP, CC or Rb) deprivation. The involvement of bcl-2 activity, established a close association between small vitamin molecules particularly DCaP, CC or Rb and the biochemical activation of apoptosis.