Telomere dysfunction: a new player in radiation sensitivity

Human individuals often exhibit important differences in their sensitivity to ionising radiation. Extensive literature links radiation sensitivity with impaired DNA repair which is due to a lack of correct functioning in many proteins involved in DNA-repair pathways and/or in DNA-damage checkpoint responses. Given that ionising radiation is an important and widespread diagnostic and therapeutic tool, it is important to investigate further those factors and mechanisms that underlie individual radiosensitivity. Recently, evidence is accumulating that telomere function may well be involved in cellular and organism responses to ionising radiation, broadening still further the currently complex and challenging scenario.     

Generation of suppressor cells by concanavalin A: a new perspective

Significantly lower mitogenic responses of fresh cells co-cultured with Con A-stimulated cells were found when compared with the responses of fresh cells co-cultured with preincubated control cells. We do not agree with the interpretation that this effect represents the generation of suppressor cells by Con A, since the responses of fresh cells cultured alone were also significantly less than when co-cultured with control cells and the same as when co-cultured with the Con A-stimulated cells. Treatment with mitomycin C was sufficient to prevent the preincubated cells from contributing to the mitogenic response of the fresh cells. The increased responses of fresh cells when co-cultured with preincubated cells seems analagous to the increased mitogenic responses of cells aged in vitro by preincubation without mitogen. This effect seems to be transferable to fresh cells in the absence of cell division. Although preincubation in the presence of Con A abrogates this effect, we do not interpret this as the generation of suppressor cells.     

LINP1 facilitates DNA damage repair through non-homologous end joining (NHEJ) pathway and subsequently decreases the sensitivity of cervical cancer cells to ionizing radiation

LncRNA in non-homologous end joining (NHEJ) pathway 1 (LINP1) is an lncRNA which promotes therapeutic resistance in triple-negative breast cancer (TNBC). However, the expression and function of LINP1 in cervical cancer is not yet well-understood. In this study, we evaluated the expression levels of LINP1 in tumor tissues and cell lines of cervical cancer. We found that LINP1 associates with NHEJ proteins (Ku80 and DNA-PKcs). LINP1 translocates from cytosol to nucleus in response to irradiation. In addition, LINP1 knockdown significantly increases the levels of cleaved caspase3 and PARP, leading to enhanced cell apoptosis after ionizing radiation (IR). LINP1-knockdown cells showed delayed repairs of DNA double-strand breaks (DSBs) after IR. Finally, LINP1 knockdown increases radiosensitivity of Hela S3 cells. These results suggest that LINP1 facilitates DSBs repair through NHEJ pathway and may thus serve as a prognostic marker and a potential target for the therapy of cervical cancer.     

The mre11 nuclease is critical for the sensitivity of cells to chk1 inhibition

The Chk1 kinase is required for the arrest of cell cycle progression when DNA is damaged, and for stabilizing stalled replication forks. As a consequence, many Chk1 inhibitors have been developed and tested for their potential to enhance DNA damage-induced tumor cell killing. However, inhibition of Chk1 alone, without any additional exogenous agent, can be cytotoxic. Understanding the underlying mechanisms of this sensitivity is critical for defining which patients might respond best to therapy with Chk1 inhibitors. We have investigated the mechanism of sensitivity in U2OS osteosarcoma cells. Upon incubation with the Chk1 inhibitor MK-8776, single-stranded DNA regions (ssDNA) and double-strand breaks (DSB) begin to appear within 6 h. These DSB have been attributed to the structure-specific DNA endonuclease, Mus81. The Mre11/Rad50/Nbs1 complex is known to be responsible for the resection of DSB to ssDNA. However, we show that inhibition of the Mre11 nuclease activity leads, not only to a decrease in the amount of ssDNA following Chk1 inhibition, but also inhibits the formation of DSB, suggesting that DSB are a consequence of ssDNA formation. These findings were corroborated by the discovery that Mre11-deficient ATLD1 cells are highly resistant to MK-8776 and form neither ssDNA nor DSB following treatment. However, once complimented with exogenous Mre11, the cells accumulate both ssDNA and DSB when incubated with MK-8776. Our findings suggest that Mre11 provides the link between aberrant activation of Cdc25A/Cdk2 and Mus81. The results highlight a novel role for Mre11 in the production of DSB and may help define which tumors are more sensitive to MK-8776 alone or in combination with DNA damaging agents.     

Colon cancer cells maintain low levels of pyruvate to avoid cell death caused by inhibition of HDAC1/HDAC3

Human colon cancer cells and primary colon cancer silence the gene coding for LDH (lactate dehydrogenase)-B and up-regulate the gene coding for LDH-A, resulting in effective conversion of pyruvate into lactate. This is associated with markedly reduced levels of pyruvate in cancer cells compared with non-malignant cells. The silencing of LDH-B in cancer cells occurs via DNA methylation, with involvement of the DNMTs (DNA methyltransferases) DNMT1 and DNMT3b. Colon cancer is also associated with the expression of pyruvate kinase M2, a splice variant with low catalytic activity. We have shown recently that pyruvate is an inhibitor of HDACs (histone deacetylases). Here we show that pyruvate is a specific inhibitor of HDAC1 and HDAC3. Lactate has no effect on any of the HDACs examined. Colon cancer cells exhibit increased HDAC activity compared with non-malignant cells. HDAC1 and HDAC3 are up-regulated in colon cancer cells and in primary colon cancer, and siRNA (small interfering RNA)-mediated silencing of HDAC1 and HDAC3 in colon cancer cells induces apoptosis. Colon cancer cells silence SLC5A8, the gene coding for a Na(+)-coupled pyruvate transporter. Heterologous expression of SLC5A8 in the human colon cancer cell line SW480 leads to inhibition of HDAC activity when cultured in the presence of pyruvate. This process is associated with an increase in intracellular levels of pyruvate, increase in the acetylation status of histone H4, and enhanced cell death. These studies show that cancer cells effectively maintain low levels of pyruvate to prevent inhibition of HDAC1/HDAC3 and thereby to evade cell death.     

DECR1 directly activates HSL to promote lipolysis in cervical cancer cells

Fatty acids have a high turnover rate in cancer cells to supply energy for tumor growth and proliferation. Lipolysis is particularly important for the regulation of fatty acid homeostasis and in the maintenance of cancer cells. In the current study, we explored how 2,4-Dienoyl-CoA reductase (DECR1), a short-chain dehydrogenase/reductase associated with mitochondrial and cytoplasmic compartments, promotes cancer cell growth. We report that DECR1 overexpression significantly reduced the triglyceride (TAG) content in HeLa cells; conversely, DECR1 silencing increased intracellular TAG content. Subsequently, our experiments demonstrate that DECR1 promotes lipolysis via effects on hormone sensitive lipase (HSL). The direct interaction of DECR1 with HSL increases HSL phosphorylation and activity, facilitating the translocation of HSL to lipid droplets. The ensuing enhancement of lipolysis thus increases the release of free fatty acids. Downstream effects include the promotion of cervical cancer cell migration and growth, associated with the enhanced levels of p62 protein. In summary, high levels of DECR1 serves to enhance lipolysis and the release of fatty acid energy stores to support cervical cancer cell growth.     

Cytoglobin promotes sensitivity to ferroptosis by regulating p53-YAP1 axis in colon cancer cells

Ferroptosis is an iron-dependent mode of non-apoptotic cell death characterized by accumulation of lipid reactive oxygen species (ROS). As a regulator of ROS, cytoglobin (CYGB) plays an important role in oxygen homeostasis and acts as a tumour suppressor. However, the mechanism by which CYGB regulates cell death is largely unknown. Here, we show that CYGB overexpression increased ROS accumulation and disrupted mitochondrial function as determined by the oxygen consumption rate and membrane potential. Importantly, ferroptotic features with accumulated lipid ROS and malondialdehyde were observed in CYGB-overexpressing colorectal cancer cells. Moreover, CYGB significantly increased the sensitivity of cancer cells to RSL3- and erastin-induced ferroptotic cell death. Mechanically, both YAP1 and p53 were significantly increased based on the RNA sequencing. The knock-down of YAP1 alleviated production of lipid ROS and sensitivity to ferroptosis in CYGB overexpressed cells. Furthermore, YAP1 was identified to be inhibited by p53 knock-down. Finally, high expression level of CYGB had the close correlation with key genes YAP1 and ACSL4 in ferroptosis pathway in colon cancer based on analysis from TCGA data. Collectively, our results demonstrated a novel tumour suppressor role of CYGB through p53-YAP1 axis in regulating ferroptosis and suggested a potential therapeutic approach for colon cancer.     

Proteasomal inhibition sensitizes cervical cancer cells to mitomycin C-induced bystander effect: the role of tumor microenvironment

Inaccessibility of drugs to poorly vascularized strata of tumor is one of the limiting factors in cancer therapy. With the advent of bystander effect (BE), it is possible to perpetuate the cellular damage from drug-exposed cells to the unexposed ones. However, the role of infiltrating tumor-associated macrophages (TAMs), an integral part of the tumor microenvironment, in further intensifying BE remains obscure. In the present study, we evaluated the effect of mitomycin C (MMC), a chemotherapeutic drug, to induce BE in cervical carcinoma. By using cervical cancer cells and differentiated macrophages, we demonstrate that MMC induces the expression of FasL via upregulation of PPARγ in both cell types (effector cells) in vitro, but it failed to induce bystander killing in cervical cancer cells. This effect was primarily owing to the proteasomal degradation of death receptors in the cervical cancer cells. Pre-treatment of cervical cancer cells with MG132, a proteasomal inhibitor, facilitates MMC-mediated bystander killing in co-culture and condition medium transfer experiments. In NOD/SCID mice bearing xenografted HeLa tumors administered with the combination of MMC and MG132, tumor progression was significantly reduced in comparison with those treated with either agent alone. FasL expression was increased in TAMs, and the enhanced level of Fas was observed in these tumor sections, thereby causing increased apoptosis. These findings suggest that restoration of death receptor-mediated apoptotic pathway in tumor cells with concomitant activation of TAMs could effectively restrict tumor growth.Owing to the heterogeneous nature and scanty vascularization, the access of anticancer regimen to all strata of the tumor is one of the major challenges in cancer therapy. Current response rate to chemotherapy is far from desirable and warrants formulating the strategies to enhance specificity and efficacy of the anticancer regimens. Of late, the phenomenon of bystander effect (BE), which refers to transmission of death signals from the drug-exposed cells to the unexposed cells, is being explored to improve the therapeutic response. Although BE has been well documented in radiotherapy^{1, 2} and experimental approaches of gene therapy,^{3, 4} very limited information is available with respect to conventional chemotherapeutic drugs. We have previously demonstrated the chemotherapy-induced bystander killing in breast cancer cells⁵ and hepatocellular carcinoma cells.⁶ Recently, other groups also have demonstrated the occurrence of chemotherapy-induced BE in breast cancer⁷ and lung cancer,^{8, 9} which is in agreement with our studies. BE has been shown to be dependent on cell type and class of drugs,⁶ and the role of tumor microenvironment in response to chemotherapeutic drug-induced BE is poorly understood.Cervical cancer is one of the most common solid tumors. Mitomycin C (MMC), a DNA alkylating agent, has been widely used in this malignancy as a constituent of combination therapy.¹⁰ From the pharmacological point of view, MMC is effective at relatively low dose with minimal organ-associated toxicity¹¹ and it has been shown to activate innate immunity.¹² However, therapeutic efficacy of MMC principally depends on other drug types in combination therapy.¹³ Therefore, a well-designed strategy that could enhance the efficacy of MMC is desirable. MMC has been demonstrated to induce BE in hepatocellular carcinoma, but not in cervical cancer cells.⁶ Although the precise mechanisms of bystander killing remain elusive, we have previously reported the involvement of death ligands,^{5, 6} which was later supported by other studies.^{7, 8, 9} The ability of cancer cells to escape programmed cell death has a critical role in the survival of cancer cells and tumor progression. Despite the presence of cellular apoptotic factors, cancer cells reprogram their molecular events and signaling to evade apoptosis.¹⁴ Moreover, it has been reported that exposure to proteasomal inhibitor inhibits the growth of various cancer cells and sensitize them to death ligand-mediated death by stabilizing death receptors.^{15, 16, 17} Considering these notions, we speculated that non-functionality of death receptors could be one of the possible factors associated with defective BE in cervical cancer. We, therefore, hypothesized that treating cervical cancer cells with combination of MMC and proteasomal inhibitor could elicit BE, and thereby may significantly improve the therapeutic outcome.Till date, studies explicate cancer cells exposed to chemotherapy as the effector cells in inducing bystander-mediated killing. However, owing to the heterogeneous nature of cellular population in tumor, other cellular components are also likely to have a key role in inducing BE. Tumor microenvironment consists of a heterogeneous mass of malignant as well as nonmalignant cells. The nonmalignant cells include endothelial, fibroblast and immune cells that establish multitude of interactions among themselves and also with malignant cells.⁹ Macrophages are the most abundant immune cells present in tumors, also termed as tumor-associated macrophages (TAMs).¹⁸ TAMs are differentiated monocytes that infiltrate the tumor microenvironment, and are exposed to chemotherapeutic regimen. Studies have demonstrated that TAMs could account for approximately more than 60% of tumor mass in some cancers.^{19, 20, 21} TAMs exposed to radiations² and chemotherapy²² have been shown to have a significant role in inducing BE. Studies support the notion that targeting TAMs could improve the therapeutic index of various drugs.^{10, 23} Increased sensitivity to cyclophosphamide¹⁴ and cisplatin²⁴ has been shown in co-culture system involving cancer cells and macrophages. Under chemotherapy, increased recruitment of macrophages with enhanced expression of tumoricidal factors like perforin and granzyme,²² death ligands¹⁰ or ROS ²⁵ has been reported in tumors. Therefore, we speculated that BE could further be amplified by infiltrating macrophages resulting in enhanced therapeutic efficacy of anticancer regimens. In the present study, we evaluated combination effect of MMC and MG132 in enhancing bystander killing of cancer cells in vitro and in vivo, in part, through the involvement of cancer cells and TAMs. Herein, we demonstrate that stabilization of Fas on cervical cancer cells facilitates dramatic reduction in tumor progression as a consequence of increase in apoptosis. This study could be helpful in designing novel therapeutic strategies in treating cancer by involving proteasomal inhibitors in combination with chemotherapeutic drugs that specifically activate death receptor-mediated killing.     

Silencing of Bmi-1 gene by RNA interference enhances sensitivity to doxorubicin in breast cancer cells

The oncogene Bmi-1 is highly up-regulated in breast carcinoma and is found to be efficient in preventing apoptosis of the cancer cells. Doxorubicin is an important chemotherapeutic agent against breast carcinoma. However, the effective therapeutic response to doxorubicin is often associated with severe toxicity. The present study is targetted at developing a strategy to increase doxorubicin sensitivity to lower doses without compromising its efficacy. A stable cell line with a persistent silencing of Bmi-1 was established. MTT assay was performed to evaluate 50% inhibitory concentration (IC50) values of doxorubicin. Apoptosis was detected by FCM and the expression of related genes [phosphor-Akt (pAkt), totle-Akt (tAkt), Bcl-2 and Bax] was studied by Western blot. In vivo, the sensitivity of the tumor tissues against doxorubicin was evaluated by transplanted MCF-7 nude mice model and the apoptosis of tissue cells was detected by TUNEL assay. The expression of pAkt and Bcl-2 was down-regulated, whereas Bax was up-regulated in Bmi-1 silencing cells. The results obtained indicated that silencing of Bmi-1 can render MCF-7 cells more sensitive to doxorubicin which induced a significantly higher percentage of apoptosis cells in vitro and in vivo. All together these results clearly demonstrate that Bmi-1 siliencing combined treatment of doxorubicin might be a new strategy for biological treatment on breast cancer.     

Hypoxanthine transport in mammalian cells: Cell type-specific differences in sensitivity to inhibition by dipyridamole and uridine

Summary We have measured by rapid kinetic techniques the zero-trans influx of hypoxanthine in various cell lines and its sensitivity to inhibition by uridine, dipyridamole, nitrobenzylthioinosine and nitrobenzylthiopurine. The results and those reported earlier divided the cells into two distinct groups. In mouse P388, L1210 and L929 cells uridine and hypoxanthine had little effect on the transport of each other, supporting the view that nucleosides and hypoxanthine are transported by different carriers. In these cells, hypoxanthine transport was also uniquely resistant to inhibition by dipyridamole (IC₅₀ (50% inhibition dose) >30M). In Novikoff and HTC rat hepatoma, Chinese hamster ovary and Ehrlich ascites tumor cells, on the other hand, hypoxanthine and uridine inhibited the transport of each other about 50% at a concentration corresponding to the Michaelis-Menten constant of their transport, and hypoxanthine transport was strongly inhibited by dipyridamole (IC₅₀=100 to 400nM). Although these results are compatible with the view that nucleosides and hypoxanthine are transported by a common carrier in these cells, this conclusion is not supported by the finding that uridine transport is strongly inhibited in some of these cell lines, as in first group of cells, by nitrobenzylthioinsine, whereas hypoxanthine transport is highly resistant in all cell lines tested. In contrast, the transport of both substrates is highly resistant to inhibition by nitrobenzylthiopurine. The Michaelis-Menten constants for uridine transport are about the same in all cell lines. The Michaelis-Menten constants for hypoxanthine transport are similar to those for uridine transport in some cell lines, but are much higher in others. This difference is unrelated to the sensitivity of uridine and hypoxanthine transport to inhibition by each other or dipyridamole.     

Changes in sensitivity of human tumour cells to growth inhibition by proteinase inhibitors.

Inhibition of a cell-surface proteinase can inhibit the growth of many normal human cell types in culture. Some tumour cells are also sensitive to proteinase inhibitors, but others are resistant, and continue to grow in the presence of these inhibitors. Here we describe two human tumour cell lines which convert from the sensitive to the resistant state. In one case, the conversion occurs during routine passaging, but, in the other, it is determined by growth conditions, and is reversible.     

Down-regulation of Notch1 by gamma-secretase inhibition contributes to cell growth inhibition and apoptosis in ovarian cancer cells A2780

The release of Notch intracellular domain (NICD) is mediated by γ-secretase. γ-Secretase inhibitors have been shown to be potent inhibitors of NICD. We hypothesized that Notch1 is acting as an oncogene in ovarian cancer and that inhibition of Notch1 would lead to inhibition of cell growth and apoptotic cell death in ovarian cancer cells. In this study, expressions of Notch1 and hes1 in four human ovarian cancer (A2780, SKOV3, HO-8910, and HO-8910PM), and one ovarian surface (IOSE 144) cell lines were detected by Western blot and quantitative real-time RT-PCR. The effects of γ-secretase inhibition (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, DAPT) were measured by MTT assay, flow cytometry, ELISA and colony-forming assay. Our results showed that Notch1 and hes1 were found in all the four human ovarian cancer and IOSE 144 cell lines, and they were significantly higher in ovarian cancer cells A2780 compared to another four ovarian cells. Down-regulation of Notch1 expression by DAPT was able to substantially inhibit cell growth, induce G1 cell cycle arrest and induce cell apoptosis in A2780 in dose- and time-dependent manner. In addition, hes1 was found to be down-regulated in dose- and time-dependent manner by DAPT in A2780. These results demonstrate that treatment with DAPT leads to growth inhibition and apoptosis of A2780 cells in dose- and time-dependent manner. These findings also support the conclusion that blocking of the Notch1 activity by γ-secretase inhibitors represents a potentially attractive strategy of targeted therapy for ovarian cancer.     

Inducible expression of maize polyamine oxidase in the nucleus of MCF-7 human breast cancer cells confers sensitivity to etoposide

Summary. In this study, polyamine oxidase from maize (MPAO), which is involved in the terminal catabolism of spermidine and spermine to produce an aminoaldehyde, 1,3-diaminopropane and H₂O₂, has been conditionally expressed at high levels in the nucleus of MCF-7 human breast cancer cells, with the aim to interfere with polyamine homeostasis and cell proliferation. Recombinant MPAO expression induced accumulation of a high amount of 1,3-diaminopropane, an increase of putrescine levels and no alteration in the cellular content of spermine and spermidine. Furthermore, recombinant MPAO expression did not interfere with cell growth of MCF-7 cells under normal conditions but it did confer higher growth sensitivity to etoposide, a DNA topoisomerase II inhibitor widely used as antineoplastic drug. These data suggest polyamine oxidases as a potential tool to improve the efficiency of antiproliferative agents despite the difficulty to interfere with cellular homeostasis of spermine and spermidine. Authors’ address: Dr. Paraskevi Tavladoraki, Department of Biology, University ‘Roma Tre’, Viale G. Marconi 446, 00146 Rome, Italy