cIAP1/2 are involved in the radiosensitizing effect of birinapant on NSCLC cell line in vitro

Tumour radioresistance is a major problem for cancer radiation therapy. To identify the underlying mechanisms of this resistance, we used human non-small cell lung cancer (NSCLC) cell lines and focused on the Inhibitor of Apoptosis Protein (IAP) family, which contributes to tumourigenesis and chemoresistance. We investigated the possible correlation between radioresistance in six NSCLC cell lines and IAP protein levels and tested the radiosensitizing effect of birinapant in vitro, a molecule that mimics the second mitochondria-derived activator of caspase. We found that birinapant-induced apoptosis and inhibited the proliferation of NSCLC cells after exposure to radiation. These effects were induced by birinapant downregulation of cIAP protein levels and changes of cIAP gene expression. Overall, birinapant can inhibit tumour growth of NSCLC cell lines to ironizing radiation and act as a promising strategy to overcome radioresistance in NSCLC.     

Radioprotective effect of 5-hydroxytryptamine and 5-hydroxytryptophan on mammalian cells irradiated in vitro.

The radioprotective effect in vitro of 5-hydroxytryptamine (5-HT) and 5-hydroxytryptophan (k-HTP) was studied with cultured mammalian cells of three cell lines: 5-HT-synthesizing FMA and 5-HT-non-synthesizing FM3A and B16-C2W. In these cells, the addition of 5-HT to the suspending medium induced only a weak protection or no protection at all. The increase in the 5-HT content of these cells at the time of irradiation was negligible. Pre-incubation of cells for 40 min in a 5-HTP-containing medium resulted in an elevation of the 5-HT content concomitantly with an increase in the radioresistance of FMA cells, where the DRF at 1 per cent was 1 x 8. In FM3A and B16-C2W cells such an effect was not observed. The same relationship between 5-HT content and radioresistance was also observed in FMA cells which were cultured in different densities or with reserpine. These results strongly suggest that the substance playing the main role in the induction of radioresistance in cells in vitro is the 5-HT that exists in the cells.     

Intracellular signaling pathways regulating radioresistance of human prostate carcinoma cells

Radiation therapy plays an important role in the management of prostate carcinoma. However, the problem of radioresistance and molecular mechanisms by which prostate carcinoma cells overcome cytotoxic effects of radiation therapy remains to be elucidated. In order to investigate possible intracellular mechanisms underlying the prostate carcinoma recurrences after radiotherapy, we have established three radiation-resistant prostate cancer cell lines, LNCaP-IRR, PC3-IRR, and Du145-IRR derived from the parental LNCaP, PC3, and Du145 prostate cancer cells by repetitive exposure to ionizing radiation. LNCaP-IRR, PC3-IRR, and Du145-IRR cells (prostate carcinoma cells recurred after radiation exposure (IRR cells)) showed higher radioresistance and cell motility than parental cell lines. IRR cells exhibited higher levels of androgen and epidermal growth factor (EGF) receptors and activation of their downstream pathways, such as Ras-mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K)-Akt and Jak-STAT. In order to define additional mechanisms involved in the radioresistance development, we determined differences in the proteome profile of parental and IRR cells using 2-D DIGE followed by computational image analysis and MS. Twenty-seven proteins were found to be modulated in all three radioresistant cell lines compared to parental cells. Identified proteins revealed capacity to interact with EGF and androgen receptors related signal transduction pathways and were involved in the regulation of intracellular routs providing cell survival, increased motility, mutagenesis, and DNA repair. Our data suggest that radioresistance development is accompanied by multiple mechanisms, including activation of cell receptors and related downstream signal transduction pathways. Identified proteins regulated in the radioresistant prostate carcinoma cells can significantly intensify activation of intracellular signaling that govern cell survival, growth, proliferation, invasion, motility, and DNA repair. In addition, such analyses may be utilized in predicting cellular response to radiotherapy.     

Cadmium induces p53-dependent apoptosis in human prostate epithelial cells

Cadmium, a widespread toxic pollutant of occupational and environmental concern, is a known human carcinogen. The prostate is a potential target for cadmium carcinogenesis, although the underlying mechanisms are still unclear. Furthermore, cadmium may induce cell death by apoptosis in various cell types, and it has been hypothesized that a key factor in cadmium-induced malignant transformation is acquisition of apoptotic resistance. We investigated the in vitro effects produced by cadmium exposure in normal or tumor cells derived from human prostate epithelium, including RWPE-1 and its cadmium-transformed derivative CTPE, the primary adenocarcinoma 22Rv1 and CWR-R1 cells and LNCaP, PC-3 and DU145 metastatic cancer cell lines. Cells were treated for 24 hours with different concentrations of CdCl(2) and apoptosis, cell cycle distribution and expression of tumor suppressor proteins were analyzed. Subsequently, cellular response to cadmium was evaluated after siRNA-mediated p53 silencing in wild type p53-expressing RWPE-1 and LNCaP cells, and after adenoviral p53 overexpression in p53-deficient DU145 and PC-3 cell lines. The cell lines exhibited different sensitivity to cadmium, and 24-hour exposure to different CdCl(2) concentrations induced dose- and cell type-dependent apoptotic response and inhibition of cell proliferation that correlated with accumulation of functional p53 and overexpression of p21 in wild type p53-expressing cell lines. On the other hand, p53 silencing was able to suppress cadmium-induced apoptosis. Our results demonstrate that cadmium can induce p53-dependent apoptosis in human prostate epithelial cells and suggest p53 mutation as a possible contributing factor for the acquisition of apoptotic resistance in cadmium prostatic carcinogenesis.     

Growth factor production by Creutzfeldt-Jakob disease cell lines.

Creutzfeldt-Jakob disease (CJD), a progressive dementia of humans, is caused by an infectious agent that is closely related to the scrapie agent of sheep. Although the molecular nature of these "unconventional" agents is still a matter of speculation and controversy, even less is known concerning the mechanism(s) of their effects on the central nervous system. To gain insight into the cellular effects of these agents, we have examined a series of cell lines derived directly from CJD-infected hamster brain or produced from nontransformed rodent lines by exposure to CJD infectious fractions in vitro. These cell lines appear transformed by a variety of criteria and secrete growth factors into the culture medium. All CJD lines produce a factor that is like alpha-transforming growth factor (alpha-TGF). Conditioned medium from these CJD lines also stimulates the synthesis of glial fibrillary acidic protein in normal astrocytic cells in vitro. This effect is mimicked by purified alpha-TGF and platelet-derived growth factors. Further study of CJD-induced growth factor production may elucidate fundamental properties of these unconventional agents.     

Cytotoxicity of zinc in vitro

The effect of zinc ions on B16 mouse melanoma lines, HeLa cells and I-221 epithelial cells was investigated in vitro in order to ascertain whether sensitivity to Zn2+ is a general feature of cells in vitro and in an attempt to elucidate the mechanism(s) of zinc cytotoxicity. The proliferation of B16, HeLa and I-221 cell lines was inhibited by 1.25 x 10(-4), 1.50 x 10(-4) and 1.50 x 10(-4) mol/l Zn2+, respectively. The free radical scavengers, methimazole and ethanol, did not suppress the toxicity of Zn2+, neither did superoxide dismutase or catalase. The addition of the chelating agent EDTA reduced the zinc cytotoxicity. It was possible to suppress the cytotoxicity of zinc by increasing the concentration of either Fe2+ or Ca2+ but not Mg2+, which suggests that a prerequisite for the toxic action of zinc is entry into cells using channels that are shared with iron or calcium. This view was supported by experiments in which transferrin intensified the cytotoxic action of zinc in serum-free medium. Another agent facilitating zinc transport, prostaglandin E2, inhibited the proliferation of the B16 melanoma cell line. There were no conspicuous differences in zinc toxicity to pigmented and unpigmented cells. The toxic effect of zinc in the cell systems studied exceeded that of iron, copper, manganese and cobalt in the same concentration range. In vitro, Zn2+ should be regarded as a dangerous cation.     

Enhancement of autophagy is a potential modality for tumors refractory to radiotherapy

Radiotherapy is a well-established treatment for cancer. However, the existence of radioresistant cells is one of the major obstacles in radiotherapy. In order to understand the mechanism of cellular radioresistance and develop more effective radiotherapy, we have established clinically relevant radioresistant (CRR) cell lines, which continue to proliferate under daily exposure to 2 Gray (Gy) of X-rays for >30 days. X-ray irradiation significantly induced autophagic cells in parental cells, which was exiguous in CRR cells, suggesting that autophagic cell death is involved in cellular radiosensitivity. An autophagy inducer, rapamycin sensitized CRR cells to the level of parental cells and suppressed cell growth. An autophagy inhibitor, 3-methyladenine induced radioresistance of parental cells. Furthermore, inhibition of autophagy by knockdown of Beclin-1 made parental cells radioresistant to acute radiation. These suggest that the suppression of autophagic cell death but not apoptosis is mainly involved in cellular radioresistance. Therefore, the enhancement of autophagy may have a considerable impact on the treatment of radioresistant tumor.     

Eukaryotic translation initiation factor 4E is a cellular target for toxicity and death due to exposure to cadmium chloride

Whether translation initiation factor 4E (eIF4E), the mRNA cap binding and rate-limiting factor required for translation, is a target for cytotoxicity and cell death induced by cadmium, a human carcinogen, was investigated. Exposure of human cell lines, HCT15, PLC/PR/5, HeLa, and Chang, to cadmium chloride resulted in cytotoxicity and cell death, and this was associated with a significant decrease in eIF4E protein levels. Similarly, specific silencing of the expression of the eIF4E gene, caused by a small interfering RNA, resulted in significant cytotoxicity and cell death. On the other hand, overexpression of the eIF4E gene was protective against the cadmium-induced cytotoxicity and cell death. Further studies revealed the absence of alterations in the eIF4E mRNA level in the cadmium-treated cells despite their decreased eIF4E protein level. In addition, exposure of cells to cadmium resulted in enhanced ubiquitination of eIF4E protein while inhibitors of proteasome activity reversed the cadmium-induced decrease of eIF4E protein. Exposure of cells to cadmium, as well as the specific silencing of eIF4E gene, also resulted in decreased cellular levels of cyclin D1, a critical cell cycle and growth regulating gene, suggesting that the observed inhibition of cyclin D1 gene expression in the cadmium-treated cells is most likely due to decreased cellular level of eIF4E. Taken together, our results demonstrate that the exposure of cells to cadmium chloride resulted in cytotoxicity and cell death due to enhanced ubiquitination and consequent proteolysis of eIF4E protein, which in turn diminished cellular levels of critical genes such as cyclin D1.     

Mutual dependence of growth modifying effects of 4-hydroxynonenal and fetal calf serum in vitro

Recently, the hypothesis has been put forward that 4-hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, contributes to the mechanism of oxygen toxicity and to the selective pressure exerted by exposure to hyperoxia. Here it has been studied whether HNE itself is involved in mechanisms that convey increased resistance of the cells to the toxicity of HNE. The following four cell lines, different in their basic biological features, were used: nonmalignant Chinese hamster lung fibroblasts V79 (established cell line), human carcinoma HeLa (established cell line), pigmented murine melanoma B16f10 (primary culture), and amelanotic murine melanoma B16BL6 (primary culture). The cells were pretreated in vitro with a toxic dose of HNE (50 μM), and afterwards the effects of a second exposure to the same dose of HNE on ³H-thymidine incorporation was examined. Cells were cultured in the absence and in the presence of fetal calf serum (FCS), because it had been shown that a growth modifying effect of HNE depends on an unknown serum factor. The results showed that, regardless of the type of cells, preculturing them with 50 μM HNE in the presence of serum changed the reactivity of the cells to added serum as well as to additional HNE treatment. Thus, HNE precultured cells incorporated less ³H-thymidine in the presence of serum than if cultured under serum-free conditions. On the other hand, HNE precultured cells became less sensitive to further HNE treatment, but only if cultured in the presence of serum. It was concluded that a toxic dose of HNE renders surviving cells more resistent to oxidative stress, possibly by forming a bioactive conjugate with a serum peptide/protein. It is supposed that the same humoral factor might be responsible for the growth modifying effects of high doses of HNE as well as for the growth inhibition in the presence of serum observed for HNE precultured cells.     

RAS-Mediated radiation resistance is not linked to MAP kinase activation in two bladder carcinoma cell lines

The expression of activated RAS oncogenes has been shown to increase radioresistance in a number of cell lines. The pathways by which RAS leads to radioresistance, however, are unknown. RAS activates several signal transduction pathways, with the RAF-MAP2K-MAP kinase pathway perhaps the best studied. MAP kinase has also been shown to be activated by radiation through this pathway. Given the important role of MAP kinase in multiple signaling events, we asked if radioresistance induced by RAS was mediated through the activation of MAPK. Cells of two human bladder carcinoma cell lines were used, one with a mutated oncogenic HRAS (T24) and other with a wild-type HRAS (RT4). The surviving fraction after exposure to 2 Gy of radiation (SF2) for the T24 cell lines was found to be 0.62, whereas that for RT4 cells was 0.40. Treatment with the farnesyl transferase inhibitor (FTI) L744,832, which inhibits RAS processing and activity, decreased the SF2 of T24 cells to 0.29, whereas the SF2 of RT4 cells remained unchanged after FTI treatment, thus demonstrating the importance of RAS activation to the radiosensitivity of cells with mutated RAS. MAP kinase activation was found to be constitutive and dependent on RAS in T24 cells, while it was inducible by radiation and was independent of RAS in RT4 cells. Treatment of both cell lines with the MAP2K inhibitor PD98059 inhibited MAPK activation; however, inhibiting MAPK activation had no effect on radiation survival of T24 or RT4 cells. These data indicate that MAPK activation does not contribute to RAS-induced radioresistance in this system.     

Inhibition of Cancer Cell Growth by Exposure to a Specific Time-Varying Electromagnetic Field Involves T-Type Calcium Channels

Electromagnetic field (EMF) exposures affect many biological systems. The reproducibility of these effects is related to the intensity, duration, frequency, and pattern of the EMF. We have shown that exposure to a specific time-varying EMF can inhibit the growth of malignant cells. Thomas-EMF is a low-intensity, frequency-modulated (25-6 Hz) EMF pattern. Daily, 1 h, exposures to Thomas-EMF inhibited the growth of malignant cell lines including B16-BL6, MDA-MB-231, MCF-7, and HeLa cells but did not affect the growth of non-malignant cells. Thomas-EMF also inhibited B16-BL6 cell proliferation in vivo. B16-BL6 cells implanted in syngeneic C57b mice and exposed daily to Thomas-EMF produced smaller tumours than in sham-treated controls. In vitro studies showed that exposure of malignant cells to Thomas-EMF for > 15 min promoted Ca²⁺ influx which could be blocked by inhibitors of voltage-gated T-type Ca²⁺ channels. Blocking Ca²⁺ uptake also blocked Thomas-EMF-dependent inhibition of cell proliferation. Exposure to Thomas-EMF delayed cell cycle progression and altered cyclin expression consistent with the decrease in cell proliferation. Non-malignant cells did not show any EMF-dependent changes in Ca²⁺ influx or cell growth. These data confirm that exposure to a specific EMF pattern can affect cellular processes and that exposure to Thomas-EMF may provide a potential anti-cancer therapy.     

Chloride intracellular channel 1 identified using proteomic analysis plays an important role in the radiosensitivity of HEp‐2 cells via reactive oxygen species production

The nature of the molecules underlying the radioresistance phenotype of laryngeal cancer cells remains to be established. We initially generated radioresistant laryngeal cancer cell lines from human HEp‐2 cells with fractionated radiation. These RR‐HEp‐2 cells and isolated clones displayed more radioresistant and anti‐apoptotic phenotypes than parental HEp‐2 cells after radiation. Characteristics of RR‐Hep‐2 cell lines were confirmed by upregulation of radioresistance‐related genes, such as epidermal growth factor receptor, Hsp90, and Bcl‐xl. Subsequently, we examined proteome changes between HEp‐2 and RR‐HEp‐2 cells and identified 16 proteins showing significantly altered expression levels. Interestingly, protein expression of chloride intracellular channel 1 (CLIC1) was markedly suppressed in RR‐HEp‐2 cells, compared with non‐irradiated control cells. Suppression of CLIC1 with an indanyloxyacetic acid‐94 or small interfering RNA led to radioresistance in HEp‐2 cells by suppressing the radiation‐induced cellular ROS level. However, ectopic overexpression of CLIC1 induced radiosensitivity in RR‐HEp‐2 cells via induction of ROS level after radiation, suggesting that the protein acts as a positive regulator of ROS production. Our results collectively indicate that suppression of CLIC1 contributes to acquisition of the radioresistance phenotype of laryngeal cancer cells via inhibition of ROS production, implying that this protein is an important candidate molecule for radiotherapy in radioresistant laryngeal cancer cells.     

Staurosporine-induced apoptosis of HPV positive and negative human cervical cancer cells from different points in the cell cycle

In the present study, we compare the sensitivity of CaSki and HeLa cells (HPV positive, wild-type p53) and C33A cells (HPV negative, mutated p53) to a protein kinase inhibitor, the staurosporine (ST). We show that ST can reversibly arrest the three cervical-derived cell lines, either in G1 or in G2/M. Beyond certain ST concentrations or/and over 24 h exposure, the cells underwent apoptosis. This process took place in G1 and G2/M for C33A and CaSki plus HeLa cell lines, respectively. By using an in vitro cell-free system, we demonstrated that cytoplasmic extracts from apoptotic cells were sufficient to induce hallmarks of programmed cell death on isolated nuclei. Moreover, we found that only G2/M cytoplasmic extracts from viable CaSki and HeLa cells supplemented with ST, triggered apoptosis while exclusively G1 cytoplasmic fractions from C33A cells were efficient. Our study describes a possible involvement of the HPV infection or/and p53 status in this different ST-induced apoptosis susceptibility.     

Induction of 103-kDa gelatinase/type IV collagenase by acidic culture conditions in mouse metastatic melanoma cell lines.

Gelatinases/type IV collagenases have been shown to be involved in tumor invasion and metastasis. In this study, we examined the effect of culture medium pH on the secretion of the gelatinases from mouse B16 melanoma cell lines and human tumor cell lines using zymography analysis. The highly metastatic clone F10 of B16 melanoma did not secrete any gelatinase in neutral culture media (pH 7.1-7.3), whereas it secreted a high level of a 103-kDa gelatinase in an initial pH range of 5.4-6.1. The addition of an excess amount of glucose into a neutral culture medium also induced the gelatinase secretion from the cells by decreasing the medium pH during incubation. The extent of the acid-induced gelatinase secretion by the B16 melanoma cell lines was in the order of BL6 greater than F10 greater than F1 much greater than the parent B16 line, in good agreement with the order of their metastatic potentials. Two human cell lines (A549 and HT1080) secreted a higher level of a 90-kDa gelatinase at pH 6.8 compared with pH 7.3. The acid-induced gelatinase secretion from B16-F10 cells was blocked by cycloheximide, indicating that the enzyme induction was due to de novo synthesis. When in vitro tumor cell invasion was assayed in Boyden chambers, B16-F10 cells incubated in an acidic medium exerted a more active migration through type IV collagen gel than those in a neutral medium. These results suggest that the acidic environment formed around tumor tissues may be an important factor in invasion and metastasis of some types of tumors.     

Roles for endocytosis and low pH in herpes simplex virus entry into HeLa and Chinese hamster ovary cells

Herpes simplex virus (HSV) infection of many cultured cells, e.g., Vero cells, can be initiated by receptor binding and pH-neutral fusion with the cell surface. Here we report that a major pathway for HSV entry into the HeLa and CHO-K1 cell lines is dependent on endocytosis and exposure to a low pH. Enveloped virions were readily detected in HeLa or receptor-expressing CHO cell vesicles by electron microscopy at <30 min postinfection. As expected, images of virus fusion with the Vero cell surface were prevalent. Treatment with energy depletion or hypertonic medium, which inhibits endocytosis, prevented uptake of HSV from the HeLa and CHO cell surface relative to uptake from the Vero cell surface. Incubation of HeLa and CHO cells with the weak base ammonium chloride or the ionophore monensin, which elevate the low pH of organelles, blocked HSV entry in a dose-dependent manner. Noncytotoxic concentrations of these agents acted at an early step during infection by HSV type 1 and 2 strains. Entry mediated by the HSV receptor HveA, nectin-1, or nectin-2 was also blocked. As analyzed by fluorescence microscopy, lysosomotropic agents such as the vacuolar H(+)-ATPase inhibitor bafilomycin A1 blocked the delivery of virus capsids to the nuclei of the HeLa and CHO cell lines but had no effect on capsid transport in Vero cells. The results suggest that HSV can utilize two distinct entry pathways, depending on the type of cell encountered.     

A potent DNA synthesis inhibitor expressed by the immortal cell line SUSM-1

We have previously reported the production of DNA synthesis inhibitor proteins by both quiescent and senescent human diploid fibroblasts. Young, proliferating fibroblasts do not produce such inhibitors, but are capable of responding to either the quiescent or senescent cell DNA synthesis inhibitors. Recently, we have analyzed the immortal cell line SUSM-1 (derived from normal liver fibroblasts following exposure to carcinogen) for inhibitory activity. We have found that SUSM-1 cells produce a factor capable of inhibiting DNA synthesis in young fibroblasts. Crude extracts prepared from SUSM-1 cells inhibit DNA synthesis in a dose-dependent manner at concentrations 10-fold lower than those of either senescent or quiescent fibroblast cell extracts. SUSM-1 cells are incapable of responding to the inhibitor they produce, as are three other immortal human cell lines tested. One immortal cell line, HeLa, does respond to the SUSM-1 inhibitor, though to a lesser degree than observed with normal young fibroblasts. One hypothesis is that the DNA synthesis inhibitor protein(s) of senescent cells plays a role in determining the finite in vitro life span of normal cells. The results reported here suggest that SUSM-1 cells may have escaped senescence through loss of a receptor or cofactor for the inhibitor protein(s).