Ribosome biogenesis rate, a parameter of sensitivity to chemotherapeutic drugs inhibiting rRNA synthesis

Insulin-like growth factor-I (IGF-I) signaling is strongly associated with cell growth and regulates the rate of synthesis of the rRNA precursor, the first and the key stage of ribosome biogenesis. In a screen for mediators of IGF-I signaling in cancer, we recently identified several ribosome-related proteins, including NEP1 (nucleolar essential protein 1) and WDR3 (WD repeat 3), whose homologues in yeast function in ribosome processing. The WDR3 gene and its locus on chromosome 1p12-13 have previously been linked with malignancy. Here we show that IGF-I induces expression of WDR3 in transformed cells. WDR3 depletion causes defects in ribosome biogenesis by affecting 18 S rRNA processing and also causes a transient down-regulation of precursor rRNA levels with moderate repression of RNA polymerase I activity. Suppression of WDR3 in cells expressing functional p53 reduced proliferation and arrested cells in the G(1) phase of the cell cycle. This was associated with activation of p53 and sequestration of MDM2 by ribosomal protein L11. Cells lacking functional p53 did not undergo cell cycle arrest upon suppression of WDR3. Overall, the data indicate that WDR3 has an essential function in 40 S ribosomal subunit synthesis and in ribosomal stress signaling to p53-mediated regulation of cell cycle progression in cancer cells.

In this study we investigated the involvement of p53 in cytotoxic T-lymphocyte (CTL)-induced tumor target cell killing mediated by the perforin/granzymes pathway. For this purpose we used a human CTL clone (LT12) that kills its autologous melanoma target cells (T1), harboring a wild type p53. We demonstrated initially that LT12 kills its T1 target in a perforin/granzymes-dependent manner. Confocal microscopy and Western blot analysis indicated that conjugate formed between LT12 and T1 resulted in rapid cytoplasmic accumulation of p53 and its activation in T1 target cells. Cytotoxic assay using recombinant granzyme B (GrB) showed that this serine protease is the predominant factor inducing such accumulation. Furthermore, RNA interference-mediated lowering of the p53 protein in T1 cells or pifithrin-alpha-induced p53-specific inhibition activity significantly decreased CTL-induced target killing mediated by CTL or recombinant GrB. This emphasizes that p53 is an important determinant in granzyme B-induced apoptosis. Our data show furthermore that when T1 cells were treated with streptolysin-O/granzyme B, specific phosphorylation of p53 at Ser-15 and Ser-37 residues was observed subsequent to the activation of the stress kinases ataxia telangiectasia mutated (ATM) and p38K. Treatment of T1 cells with pifithrin-alpha resulted in inhibition of p53 phosphorylation at these residues and in a significant decrease in GrB-induced apoptotic T1 cell death. Furthermore, small interference RNAs targeting p53 was also accompanied by an inhibition of streptolysin-O/granzyme B-induced apoptotic T1 cell death. The present study supports p53 induction after CTL-induced stress in target cells. These findings provide new insight into a potential role of p53 as a component involved in the dynamic regulation of the major pathway of CTL-mediated cell death and may have therapeutic implications.

Many drugs currently used in chemotherapy work by hindering the process of ribosome biogenesis. In tumors with functional p53, the inhibition of ribosome biogenesis may contribute to the efficacy of this treatment by inducing p53 stabilization. As the level of stabilized p53 is critical for the induction of cytotoxic effects, it seems useful to highlight those cancer cell characteristics that can predict the degree of p53 stabilization following the treatment with inhibitors of ribosome biogenesis. In the present study we exposed a series of p53 wild-type human cancer cell lines to drugs such as actinomycin D (ActD), doxorubicin, 5-fluorouracil and CX-5461, which hinder ribosomal RNA (rRNA) synthesis. We found that the amount of stabilized p53 was directly related to the level of ribosome biogenesis in cells before the drug treatment. This was due to different levels of inactivation of the ribosomal proteins-MDM2 pathway of p53 digestion. Inhibition of rRNA synthesis always caused cell cycle arrest, independent of the ribosome biogenesis rate of the cells, whereas apoptosis occurred only in cells with a high rDNA transcription rate. The level of p53 stabilization induced by drugs acting in different ways from the inhibition of ribosome biogenesis, such as hydroxyurea (HU) and nutlin-3, was independent of the level of ribosome biogenesis in cells and always lower than that occurring after the inhibition of rRNA synthesis. Interestingly, in cells with a low ribosome biogenesis rate, the combined treatment with ActD and HU exerted an additive effect on p53 stabilization. These results indicated that (i) drugs inhibiting ribosome biogenesis may be highly effective in p53 wild-type cancers with a high ribosome biogenesis rate, as they induce apoptotic cell death, and (ii) the combination of drugs capable of stabilizing p53 through different mechanisms may be useful for treating cancers with a low ribosome biogenesis rate.

In this study, endogenous long chain ceramides were measured in 32 human head and neck squamous cell carcinoma (HNSCC) and 10 nonsquamous head and neck carcinoma tumor tissues, as compared with adjacent noncancerous tissues, by liquid chromatography/mass spectroscopy. Interestingly, only one specific ceramide, C(18:0)-ceramide, was selectively down-regulated in the majority of HNSCC tumor tissues. On the other hand, in nonsquamous tumor tissues, this selectivity for C18-ceramide was not detected. These data suggested the hypotheses that decreased levels of C18-ceramide might impart a growth advantage to HNSCC cells and that increased generation of C18-ceramide may be involved in the inhibition of growth. These roles were examined by reconstitution of C18-ceramide at physiologically relevant concentrations in UM-SCC-22A cells (squamous cell carcinoma of hypopharynx) via overexpression of mammalian upstream regulator of growth and differentiation factor 1 (mUOG1), a mouse homologue of longevity assurance gene 1 (mLAG1), which has been shown to specifically induce the generation of C18-ceramide. Liquid chromatography/mass spectroscopy analysis showed that overexpression of the mLAG1/mUOG1 resulted in increased levels of only C(18:0)-ceramide by approximately 2-fold, i.e. concentrations similar to those of normal head and neck tissues. Importantly, increased generation of C18-ceramide by mLAG1/mUOG1 inhibited cell growth (approximately 70-80%), which mechanistically involved the modulation of telomerase activity and induction of apoptotic cell death by mitochondrial dysfunction. In conclusion, this study demonstrates, for the first time, a biological role for LAG1 and C18-ceramide in the regulation of growth of HNSCC.

Ribosome biogenesis provides the molecular machinery required for protein synthesis, thus it dictates the ability of a cell to grow and proliferate. We have recently demonstrated that inhibiting ribosome biogenesis with a small molecule that inhibits RNA Polymerase (POL) I driven transcription (CX5461) selectively killed B-lymphoma cells in vivo while maintaining a viable wild-type B cell population (Bywater et al., Cancer Cell 22: p51, 2012). The therapeutic effect was mediated via nucleolar disruption and activation of p53-dependent apoptotic signalling. Although this response was dependent on p53, cell lines from solid tumors with mutant p53 also respond to CX5461 (Drygin et al., Cancer Res 71: p1418, 2011). Dysregulation of both the PI3K and RAS/ERK pathways are common in ovarian cancer and BRAF is mutated and activated in over 50% of melanomas; a cancer that also commonly expresses wild type-p53. Given that both these pathways are critical regulators of ribosome biogenesis, we hypothesized that targeting ribosome biogenesis may be a new and potent approach to treating these cancers. To determine the efficacy of CX5461 a panel of ovarian cancer and melanoma cell lines were treated with CX5461 and cell proliferation assessed. The majority of cell lines were sensitive to CX5461 and differential gene expression between resistant and sensitive ovarian cancer cell lines revealed sensitivity was associated with signatures of increased MYC signalling and BRCA1 and 2 mutation. Gene expression and assessment of rDNA transcription suggested that resistance in both ovarian cancer and melanoma cell lines was associated with an inability of CX5461 to inhibit POL1 driven transcription. These studies indicate that targeting ribosome biogenesis and function may provide a new therapeutic option for treating ovarian cancer and melanoma. Citation Format: Karen E. Sheppard, Natalie Brajanovski, Katherine M. Hannan, Jessica Ahearn, Jason Ellul, Denis Drygin, Sean O'Brien, Grant McArthur, Ross D. Hannan, Richard B. Pearson. Targeting ribosome biogenesis with CX5461 as a potential treatment for melanoma and ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2718. doi:10.1158/1538-7445.AM2014-2718

In the present study, we aimed to investigate the anticancer properties of Theracurmin®, a novel form of the yellow curry pigment curcumin, as well as explore the molecular mechanisms of the potential anticancer effects of Theracurmin® on human prostate cancer and bladder cancer cells in vitro. The proliferation of cancer cells was examined by using the Cell Counting Kit-8. The clonogenic growth potential was determined by clonogenic assay. Cell cycle distribution was evaluated by flow cytometry using propidium iodide staining. Western blot analysis was applied to explore the expression patterns of molecules associated with apoptotic cell death and cell cycle checkpoint. We noted that Theracurmin® and curcumin exhibited similar anticancer effects in both androgen-dependent and -independent human prostate cancer cells in a dose- and time-dependent manner. These agents reduced cell viability and clonogenic growth potential by inducing apoptosis and cell cycle disturbance in human prostate cancer cells. Theracurmin® and curcumin also exerted marked anticancer effects on human bladder cancer cells, even in cisplatin-resistant T24R2 cells, in a dose- and time-dependent manner. Moreover, Theracurmin® and curcumin treatment decreased cell viability and clonogenicity via induction of apoptotic cell death and cell cycle dysregulation in human bladder cancer cells. In conclusion, our study suggests that Theracurmin® has potential as an anticancer agent in complementary and alternative medicine for these urological cancers.

Mistletoe lectin has been reported to induce apoptosis in different cancer cell lines in vitro and to show antitumor activity against a variety of tumors in animal models. We previously demonstrated the Korean mistletoe lectin (Viscum album var. coloratum, VCA)-induced apoptosis by down-regulation of Bcl-2 and telomerase activity and by up-regulation of Bax through p53- and p21-independent pathway in hepatoma cells. In the present study, we observed the induction of apoptotic cell death through activation of caspase-3 and the inhibition of telomerase activity through transcriptional down-regulation of hTERT in the VCA-treated A253 cells. We also observed the inhibition of telomerase activity and induction of apoptosis resulted from dephosphorylation of Akt in the survival signaling pathways. In addition, combining VCA with the inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) upstream of Akt, wortmannin and LY294002 showed an additive inhibitory effect of telomerase activity. In contrast, the inhibitor of protein phosphatase 2A (PP2A), okadaic acid inhibited VCA-induced dephosphorylation of Akt and inhibition of telomerase activity. Taken together, VCA induces apoptotic cell death through Akt signaling pathway in correlated with the inhibition of telomerase activity and the activation of caspase-3. From these results, together with our previous studies, we suggest that VCA triggers molecular changes that resulting in the inhibition of cell growth and the induction of apoptotic cell death of cancer cells, which suggest that VCA may be useful as chemotherapeutic agent for cancer cells.

Cancer is characterized by hyperactivation of ribosome biogenesis which depends on increased RNA Polymerase I (Pol I) transcription. Inhibition of Pol I transcription causes nucleolar stress that leads to the release of ribosomal proteins from the nucleolus into the nucleoplasm where they can sequester the p53 inhibitory protein MDM2, causing activation of p53 and induction of apoptosis. The inhibition of Pol I transcription as a therapeutic approach is significant because it impacts two critically balanced processes, proliferation and apoptosis, that regulate cancer cell survival. CX-5461 is a potent and selective inhibitor of Pol I transcription. CX-5461 acts at the initiation stage of Pol I transcription through the disruption of the SL1/rDNA complex. CX-5461 is non-genotoxic and does not inhibit DNA replication, protein translation or RNA Polymerase II transcription. We have previously demonstrated that CX-5461 triggers autophagic cell death in solid tumor cell lines and exhibits antitumor activity in xenograft models, highlighting the importance of Pol I transcription in cancer (Drygin et al. Cancer Res. in press). In preparation for clinical testing we sought to identify the most sensitive indications by evaluating CX-5461 against a panel of genetically diverse cancer cell lines. CX-5461 exhibited a broad range of antiproliferative activity with wild-type (wt) p53 cells derived from hematological malignancies being the most sensitive (median IC50 = 5 nM). Other cell types, i.e. p53 mutated hematological, p53wt and p53 mutated solid tumors were less sensitive to CX-5461 (median IC50s = 94, 164 and 265 nM respectively). In contrast, the median IC50 against normal cells was 5 uM indicating that CX-5461 selectively kills cancer cells. Further molecular characterization revealed that CX-5461 treatment of p53wt hematologic cancer cells inhibited rRNA synthesis, stabilized p53, activated p21, caused cell cycle arrest and induced apoptosis. Chemical inhibition of p53 prevented the induction of apoptosis indicating that CX-5461 acts through activation of p53. Interestingly, while p53 mutations impacted the activity of CX-5461, other genetic alterations known to silence p53 response, i.e. deletion of ARF, did not affect sensitivity to CX-5461 (Bywater et al. 2010 AACR Ann Met Proceedings). Activation of p53 has long been considered an attractive approach for treating cancers because of the surveillance function of p53 to remove abnormal cells via induction of apoptotic cell death. The fact that mutations or deletions of the p53 gene are relatively rare in hematological malignancies, coupled with our data that p53wt hematologic cancer cells are particularly sensitive to CX-5461 provides compelling rationale for evaluating CX-5461 in this patient population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4511. doi:10.1158/1538-7445.AM2011-4511

JAK2 plays important roles in the regulation of a variety of cellular processes including cell migration, proliferation, and protection from apoptosis. Recently the L611S point mutation in JAK2 has been identified in a child with acute lymphoblastic leukemia. Here we analyzed the mechanism by which JAK2 exhibits its oncogenicity. In BaF3 murine hematopoietic cells, L611S mutant increased the expression of antiapoptotic proteins including X chromosome-linked inhibitor of apoptosis protein, inhibitor of apoptosis protein, and Bcl-XL. We also showed that JAK2 L611S mutant protects BaF3 cells from cytokine withdrawal-induced apoptotic cell death and leads to cytokine-independent cell growth. Furthermore BaF3 cells expressing JAK2 L611S mutant gained the ability to induce tumorigenesis in nude mice. The L611S mutant also exhibited malignancy, including prompt invasion and spreading into various organs, leading to rapid lethality of the mice. Finally we showed that a specific JAK2 inhibitor, AG490, potently inhibited cytokine-independent cell growth induced by JAK2 L611S mutant via the induction of apoptotic cell death. In addition, treatment with AG490 significantly inhibited the JAK2 L611S mutant-induced tumorigenesis in nude mice. Thus, our results both in vitro and in vivo strongly suggest that L611S mutant of JAK2 harbors potent oncogenic activity, and this probably requires the antiapoptotic signaling pathway.

Selective induction of apoptotic cancer cell death by curcumin-loaded PEGylated lipid nanoparticles.

Bee venom has been used as a traditional medicine to treat arthritis, rheumatism, back pain, cancerous tumors, and skin diseases. However, the effects of bee venom on the prostate cancer and their action mechanisms have not been reported yet. To determine the effect of bee venom and its major component, melittin on the prostate cancer cells, apoptosis is analyzed by tunnel assay and apoptotic gene expression. For xenograft studies, bee venom was administrated intraperitoneally twice per week for 4 weeks, and the tumor growth was measured and the tumor were analyzed by immunohistochemistry. To investigate whether bee venom and melittin can inactivate nuclear factor kappa B (NF-κB), we assessed NF-κB activity in vitro and in vivo. Bee venom (1-10 µg/ml) and melittin (0.5-2.5 µg/ml) inhibited cancer cell growth through induction of apoptotic cell death in LNCaP, DU145, and PC-3 human prostate cancer cells. These effects were mediated by the suppression of constitutively activated NF-κB. Bee venom and melittin decreased anti-apoptotic proteins but induced pro-apoptotic proteins. However, pan caspase inhibitor abolished bee venom and melittin-induced apoptotic cell death and NF-κB inactivation. Bee venom (3-6 mg/kg) administration to nude mice implanted with PC-3 cells resulted in inhibition of tumor growth and activity of NF-κB accompanied with apoptotic cell death. Therefore, these results indicated that bee venom and melittin could inhibit prostate cancer in in vitro and in vivo, and these effects may be related to NF-κB/caspase signal mediated induction of apoptotic cell death.

Apoptosis: A barrier against cancer no more?

Several microscopy methods have been developed to assess the morphological changes in cells in the investigations of the mode of cell death in response to a stimulus. Our recent finding on the treatment of the IC50 concentration (26.67 μg/mL) of Polyalthia longifolia leaf extract indicated the induction of apoptotic cell death via the regulation of miRNA in HeLa cells. Hence, the current study was conducted to validate the function of these downregulated microRNAs in P. longifolia-treated HeLa cells using microscopic approaches. These include scanning electron microscope (SEM), transmission electron microscope (TEM), and acridine orange/propidium iodide (AO/PI)-based fluorescent microscopy techniques by observing the morphological alterations to cells after transfection with mimic miRNA. Interestingly, the morphological changes observed in this study demonstrated the apoptotic hallmarks, for instance, cell blebbing, cell shrinkage, cytoplasmic and nuclear condensation, vacuolization, cytoplasmic extrusion, and the formation of apoptotic bodies, which proved the role of dysregulated miRNAs in apoptotic HeLa cell death after treatment with the P. longifolia leaf extract. Conclusively, the current study proved the crucial role of downregulated miR-484 and miR-221-5p in the induction of apoptotic cell death in P. longifolia-treated HeLa cells using three approaches-SEM, TEM, and AO/PI-based fluorescent microscope.

Metastatic colorectal cancer responds poorly to treatment and is a leading cause of cancer related deaths. Worldwide, chemotherapy of metastatic colorectal cancer remains plagued by poor efficacy, development of resistance and serious adverse effects. Copper-imidazo[1,2-a]pyridines were previously shown by our group to be selectively active against several cancer cell lines, with three complexes, JD46(27), JD47(29), and JD88(21), showing IC50 values between 0.8 and 1.8μM against HT-29 cells. Here, we report thattreatment with the copper complexes resulted in fragmented nuclei suggestive of apoptotic cell death, which was confirmed by increased annexin V binding and caspase-3/7 activity. The copper complexes caused a loss of mitochondrial membrane potential and increased caspase-9 activity. The absence of caspase-8 activity indicated activation of the intrinsic pathway. Proteomic analysis revealed that copper-imidazo[1,2-a]pyridines decreased the expression of phosphorylated forms of p53 [phospho-p53(S15), phospho-p53(S46) and phospho-p53(S392)]. The expression of inhibitor of apoptosis proteins, XIAP, cIAP1, livin, and the antiapoptotic proteins, Bcl-2 and Bcl-x, was decreased. HO/HMOX/HSP32, expression was notably increased, which suggested the accumulation of reactive oxygen species. Increased expression of TRAIL-R2/DR5 death receptor indicated the possible dual activation of both the extrinsic and intrinsic apoptotic pathways; however, caspase-8 activation could not be demonstrated. In conclusion, the copper-imidazo[1,2-a]pyridines were effective inducers of apoptotic cell death at low micromolar concentrations and changed the expression levels of proteins important for cell survival and cell death. These copper complexes may be useful tools to better understand the complexity of signalling networks in cancer cell death in response to cell stress.

Highlights from Recent Cancer Literature