Autophagy induced by ionizing radiation promotes cell death over survival in human colorectal cancer cells

Autophagy is commonly described as a cell survival mechanism and has been implicated in chemo- and radioresistance of cancer cells. Whether ionizing radiation induced autophagy triggers tumor cell survival or cell death still remains unclear. In this study the autophagy related proteins Beclin1 and ATG7 were tested as potential targets to sensitize colorectal carcinoma cells to ionizing radiation under normoxic, hypoxic and starvation conditions. Colony formation, apoptosis and cell cycle analysis revealed that knockdown of Beclin1 or ATG7 does not enhance radiosensitivity in HCT-116 cells. Furthermore, ATG7 knockdown led to an increased survival fraction under oxygen and glutamine starvation, indicating that ionizing radiation indeed induces autophagy which, however, leads to cell death finally. These results highlight that inhibition of autophagic pathways does not generally increase therapy success but may also lead to an unfavorable outcome especially under amino acid and oxygen restriction.     

NFBD1/MDC1 is a protein of oncogenic potential in human cervical cancer

A large nuclear protein of 2089 amino acids, NFBD1/MDC1 has recently been implicated in tumorigenesis and tumor growth. In this study, we investigated its expression in cervical cancers and explored its function using gene knockdown approaches. We report here that NFBD1 expression is substantial increased in 24 of 39 cases (61.5%) of cervical cancer tissues at the mRNA level and in 35 of 60 cases (58.3%) at the protein level compared with the case matched normal tissues. Tumors with higher grade of malignancy tend to have higher levels of NFBD1 expression. By infecting cells with retroviruses expressing NFBD1 shRNA, we successfully knocked down NFBD1 expression in cervical cancer cell lines HeLa, SiHa, and CaSki. NFBD1 knockdown cells display significant growth inhibition, cell cycle arrest, higher apoptotic rate, and enhanced sensitivity to adriamycin. Furthermore, NFBD1 knockdown also inhibits the growth of HeLa cells in nude mice. Western blot analyses further revealed that NFBD1 knockdown induced Bax, Puma, and Noxa while down-regulating Bcl-2; it also up-regulated cytochrome C and activated caspases 3 and 9. Therefore, the function of NFBD1 may be involved in the CDC25C-CyclinB1/CDC2 pathway at the G2/M checkpoint, and the cytochrome C/caspase 3 apoptotic pathway. Since expression of NFBD1 seems to be related to the oncogenic potential of cervical cancer, and suppression of its expression can inhibit cancer cell growth both in vitro and in vivo, NFBD1 may be a potential therapeutic target in human cervical cancer.     

BF12, a Novel Benzofuran,Exhibits Antitumor Activity by Inhibiting Microtubules and the PI3K/Akt/mTOR Signaling Pathway in Human Cervical Cancer Cells

BF12 [(2E)‐3‐[6‐Methoxy‐2‐(3,4,5‐trimethoxybenzoyl)‐1‐benzofuran‐5‐yl]prop‐2‐enoic acid], a novel derivative of combretastatin A‐4 (CA‐4), was previously found to inhibit tumor cell lines, with a particularly strong inhibitory effect on cervical cancer cells. In this study, we investigated the microtubule polymerization effects and apoptosis signaling mechanism of BF12. BF12 showed a potent efficiency against cervical cancer cells, SiHa and HeLa, with IC₅₀ values of 1.10 and 1.06 μm , respectively. The cellular mechanism studies revealed that BF12 induced G2/M phase arrest and apoptosis in SiHa and HeLa cells, which were associated with alterations in the expression of the cell G2/M cycle checkpoint‐related proteins (cyclin B1 and cdc2) and alterations in the levels of apoptosis‐related proteins (P53, caspase‐3, Bcl‐2, and Bax) of these cells, respectively. Western blot analysis showed that BF12 inhibited the PI3 K/Akt/mTOR signaling pathway and induced apoptosis in human cervical cancer cells. BF12 was identified as a tubulin polymerization inhibitor, evidenced by the effective inhibition of tubulin polymerization and heavily disrupted microtubule networks in living SiHa and HeLa cells. By inhibiting the PI3 K/Akt/mTOR signaling pathway and inducing apoptosis in human cervical cancer cells, BF12 shows promise for use as a microtubule inhibitor.     

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.     

Suppressor of Cytokine Signaling (SOCS) Genes Are Silenced by DNA Hypermethylation and Histone Deacetylation and Regulate Response to Radiotherapy in Cervical Cancer Cells

Suppressor of cytokine signaling (SOCS) family is an important negative regulator of cytokine signaling and deregulation of SOCS has been involved in many types of cancer. All cervical cancer cell lines tested showed lower expression of SOCS1, SOCS3, and SOCS5 than normal tissue or cell lines. The immunohistochemistry result for SOCS proteins in human cervical tissue also confirmed that normal tissue expressed higher level of SOCS proteins than neighboring tumor. Similar to the regulation of SOCS in other types of cancer, DNA methylation contributed to SOCS1 downregulation in CaSki, ME-180, and HeLa cells. However, the expression of SOCS3 or SOCS5 was not recovered by the inhibition of DNA methylation. Histone deacetylation may be another regulatory mechanism involved in SOCS1 and SOCS3 expression, however, SOCS5 expression was neither affected by DNA methylation nor histone deacetylation. Ectopic expression of SOCS1 or SOCS3 conferred radioresistance to HeLa cells, which implied SOCS signaling regulates the response to radiation in cervical cancer. In this study, we have shown that SOCS expression repressed by, in part, epigenetically and altered SOCS1 and SOCS3 expression could contribute to the radiosensitive phenotype in cervical cancer.     

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.     

A novel radioresistant mechanism of galectin-1 mediated by H-Ras-dependent pathways in cervical cancer cells

Galectin-1 is a lectin recognized by galactoside-containing glycoproteins, and is involved in cancer progression and metastasis. The role of galectin-1 in radiosensitivity has not previously been investigated. Therefore, this study tests whether galectin-1 is involved in the radiosensitivity mediated by the H-Ras signaling pathway using cervical carcinoma cell lines. A knockdown of galectin-1 expression in HeLa cells decreased clonogenic survival following irradiation. The clonogenic survival increased in both HeLa and C33A cells with galectin-1 overexpression. The overexpression or knockdown of galectin-1 did not alter radiosensitivity, whereas H-Ras was silenced in both cell lines. Whereas K-Ras was knocked down, galectin-1 restored the radiosensitivity in HeLa cells and C33A cells. The knockdown of galectin-1 increased the high-dose radiation-induced cell death of HeLa cells transfected by constitutively active H-Ras. The knockdown of galectin-1 inhibited the radiation-induced phosphorylation of Raf-1 and ERK in HeLa cells. Overexpression of galectin-1 enhanced the phosphorylation of Raf-1 and ERK in C33A cells following irradiation. Galectin-1 decreased the DNA damage detected using comet assay and γ-H2AX in both cells following irradiation. These findings suggest that galectin-1 mediates radioresistance through the H-Ras-dependent pathway involved in DNA damage repair.     

Cellular and molecular mechanisms of silibinin induces cell-cycle arrest and apoptosis on HeLa cells

Silibinin, an effective anti-cancer and chemopreventive agent in various epithelial cancer models, has been reported to inhibit cancer cell growth through mitogenic signalling pathways including cervical cancer. However, the underlying mechanisms are still not well elucidated. Here, we assessed the effect of silibinin on human cervical carcinoma cell cycle modulation, apoptosis induction and associated molecular alterations by employing HeLa cell line. Silibinin treatment of HeLa cells resulted in a G2 arrest and induced a decrease in cyclin-dependent kinases involved in both G1 and G2 progression. In addition, silibinin showed a dose-dependent and a time-dependent apoptotic death in HeLa cells in both the mitochondrial pathway and the death receptor-mediated pathway, providing a strong rationale for future studies evaluating preventive and/or intervention strategies for silibinin in cervical cancer pre-clinical models.     

Loss of macrophage migration inhibitory factor impairs the growth properties of human HeLa cervical cancer cells

Objectives: This study aims to determine the role of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with cell proliferation and tumour growth in vivo. Materials and methods: Our team used RNA interference technology to knock down MIF expression in human HeLa cervical cancer cells and to establish a stable cell line lacking MIF function. Results: Our results showed that long‐term loss of MIF had little effect on cell morphology, but significantly inhibited their population growth and proliferation. The HeLa MIF‐knockdown cells retained normal apoptotic signalling pathways in response to TNF‐alpha treatment; however, they exhibited unique DNA profiles following doxorubicin treatment, suggesting that MIF may regulate a cell cycle checkpoint upon DNA damage. Our data also showed that knockdown of MIF expression in HeLa cells led to increased cell adhesion and therefore impaired their migratory capacity. More importantly, cells lacking MIF failed to either proliferate in soft agar or form tumours in vivo, when administered to nude mice. Conclusion: MIF plays a pivotal role in proliferation and tumourigenesis of human HeLa cervical carcinoma cells, and may represent a promising therapeutic target for cancer intervention.     

mTOR inhibitors radiosensitize PTEN‐deficient non‐small‐cell lung cancer cells harboring an EGFR activating mutation by inducing autophagy

Clinical resistance to gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), in patients with lung cancer has been linked to acquisition of the T790M resistance mutation in activated EGFR or amplification of MET. Phosphatase and tensin homolog (PTEN) loss has been recently reported as a gefitinib resistance mechanism in lung cancer. The aim of this study was to evaluate the efficacy of radiotherapy in non‐small‐cell lung cancer (NSCLC) with acquired gefitinib resistance caused by PTEN deficiency to suggest radiotherapy as an alternative to EGFR TKIs. PTEN deficient‐mediated gefitinib resistance was generated in HCC827 cells, an EGFR TKI sensitive NSCLC cell line, by PTEN knockdown with a lentiviral vector expressing short hairpin RNA‐targeting PTEN. The impact of PTEN knockdown on sensitivity to radiation in the presence or absence of PTEN downstream signaling inhibitors was investigated. PTEN knockdown conferred acquired resistance not only to gefitinib but also to radiation on HCC827 cells. mTOR inhibitors alone failed to reduce HCC827 cell viability, regardless of PTEN expression, but ameliorated PTEN knockdown‐induced radioresistance. PTEN knockdown‐mediated radioresistance was accompanied by repression of radiation‐induced cytotoxic autophagy, and treatment with mTOR inhibitors released the repression of cytotoxic autophagy to overcome PTEN knockdown‐induced radioresistance in HCC827 cells. These results suggest that inhibiting mTOR signaling could be an effective strategy to radiosensitize NSCLC harboring the EGFR activating mutation that acquires resistance to both TKIs and radiotherapy due to PTEN loss or inactivation mutations. J. Cell. Biochem. 114: 1248–1256, 2013. © 2012 Wiley Periodicals, Inc.     

Lethality of PAK3 and SGK2 shRNAs to Human Papillomavirus Positive Cervical Cancer Cells Is Independent of PAK3 and SGK2 Knockdown

The p21-activated kinase 3 (PAK3) and the serum and glucocorticoid-induced kinase 2 (SGK2) have been previously proposed as essential kinases for human papillomavirus positive (HPV+) cervical cancer cell survival. This was established using a shRNA knockdown approach. To validate PAK3 and SGK2 as potential targets for HPV+ cervical cancer therapy, the relationship between shRNA-induced phenotypes in HPV+ cervical cancer cells and PAK3 or SGK2 knockdown was carefully examined. We observed that the phenotypes of HPV+ cervical cancer cells induced by various PAK3 and SGK2 shRNAs could not be rescued by complement expression of respective cDNA constructs. A knockdown-deficient PAK3 shRNA with a single mismatch was sufficient to inhibit HeLa cell growth to a similar extent as wild-type PAK3 shRNA. The HPV+ cervical cancer cells were also susceptible to several non-human target shRNAs. The discrepancy between PAK3 and SGK2 shRNA-induced apoptosis and gene expression knockdown, as well as cell death stimulation, suggested that these shRNAs killed HeLa cells through different pathways that may not be target-specific. These data demonstrated that HPV+ cervical cancer cell death was not associated with RNAi-induced PAK3 and SGK2 knockdown but likely through off-target effects.     

Mechanisms of arsenic trioxide induced apoptosis of human cervical cancer HeLa cells and protection by Bcl-2

It was recently reported that arsenic trioxide (As_2O_3) can induce complete remission in patients with acute promyelocytic leukemia (APL). In this present article, the biological effect of As_2O_3 on human cervical cancer HeLa cells and HeLa cells overexpressing Bcl-2 is studied. By MTT and colony forming ability assays, morphology alteration, flow cytometric analysis, DNA gel electrephoresis and in situ cell death detection (TUNEL), it was found that As_2O_3 inhibited the growth of HeLa cells and induced G2/M arrest and apoptosis of the cells. RT-PCR, Northern blot, Western blot analysis revealed that As_2O_3 induced HeLa cell apoptosis possibly via decreasing the expression of c-myc and viral genes. HeLa cells overexpressing Bcl-2 partly resist As_2O_3 induced apoptosis, which might be relative to preventing the cells from As_2O_3 caused G2/M block, downregulation of c-myc gene expression and inhibition of viral gene expression was also noted, However, it was found that As_2O_3 at a high concentratio     

Silencing CDK4 radiosensitizes breast cancer cells by promoting apoptosis

Background

The discovery of molecular markers associated with various breast cancer subtypes has greatly improved the treatment and outcome of breast cancer patients. Unfortunately, breast cancer cells acquire resistance to various therapies. Mounting evidence suggests that resistance is rooted in the deregulation of the G1 phase regulatory machinery.

Methods

To address whether deregulation of the G1 phase regulatory machinery contributes to radiotherapy resistance, the MCF10A immortalized human mammary epithelial cell line, ER-PR-Her2+ and ER-PR-Her2- breast cancer cell lines were irradiated. Colony formation assays measured radioresistance, while immunocytochemistry, Western blots, and flow cytometry measured the cell cycle, DNA replication, mitosis, apoptosis, and DNA breaks.

Results

Molecular markers common to all cell lines were overexpressed, including cyclin A1 and cyclin D1, which impinge on CDK2 and CDK4 activities, respectively. We addressed their potential role in radioresistance by generating cell lines stably expressing small hairpin RNAs (shRNA) against CDK2 and CDK4. None of the cell lines knocked down for CDK2 displayed radiosensitization. In contrast, all cell lines knocked down for CDK4 were significantly radiosensitized, and a CDK4/CDK6 inhibitor sensitized MDA-MB-468 to radiation induced apoptosis. Our data showed that silencing CDK4 significantly increases radiation induced cell apoptosis in cell lines without significantly altering cell cycle progression, or DNA repair after irradiation. Our results indicate lower levels of phospho-Bad at ser136 upon CDK4 silencing and ionizing radiation, which has been shown to signal apoptosis.

Conclusion

Based on our data we conclude that knockdown of CDK4 activity sensitizes breast cancer cells to radiation by activating apoptosis pathways.

     

The farnesyltransferase inhibitor FTI-277 suppresses the 24-kDa FGF2-induced radioresistance in HeLa cells expressing wild-type RAS.

In this paper, we describe the effect of the inhibitor of farnesyltransferase (FTI-277) on radioresistance induced by the 24-kDa isoform of FGF2 in human cells expressing wild-type RAS. Treatment with FTI-277 (20 microM) for 48 h prior to irradiation led to a significant decrease in survival of radioresistant cells expressing the 24-kDa isoform (HeLa 3A) but had no effect on the survival of control cells (HeLa PINA). The radiosensitizing effect of FTI-277 is accompanied by a stimulation of postmitotic cell death in HeLa 3A cells and by a reduction in G(2)/M-phase arrest in both cell types. These results clearly demonstrate that at least one farnesylated protein is involved in the regulation of the radioresistance induced by the 24-kDa isoform of FGF2. Furthermore, the radiation-induced G(2)/M-phase arrest is also under the control of farnesylated protein. This work also demonstrates that FTase inhibitors may be effective radiosensitizers of certain human tumors with wild-type RAS.     

Ubiquitin B在HSP90抑制剂17-AAG诱导人宫颈癌HeLa细胞周期阻滞中的作用

目的 研究Ubiquitin B（Ubb）在热休克蛋白90（HSP90）抑制剂17-AAG诱导人宫颈癌HeLa细胞周期阻滞中的作用及机制.方法 不同浓度17-AAG处理HeLa细胞后,流式细胞术检测细胞周期分布,荧光分光光度法检测细胞蛋白酶体活性变化;Ubb siRNA 转染HeLa细胞后,Real Time PCR法检测Ubb干扰效应,Western 印迹检测细胞周期相关蛋白的表达改变.结果 17-AAG可以诱导HeLa细胞阻滞于G2/M期,同时显著增强细胞内糜蛋白酶样蛋白酶体活性,并且两者的变化均呈现剂量依赖性;干扰HeLa细胞内Ubb后,可以逆转17-AAG引起的G2/M期阻滞;17-AAG可明显下调HeLa细胞周期相关蛋白Cdk1和Hec1的表达,并且这一变化也是Ubb依赖的.结论 Ubb在17-AAG诱导的HeLa细胞周期阻滞中发挥重要作用,Ubb和HSP90抑制剂17-AAG在功能上相互关联,可能成为宫颈癌治疗的新靶点.     

3-(3-Methoxyphenyl)-6-(3-amino-4-methoxyphenyl)-7H-[1,2,4] triazolo [3,4-b][1,3,4] thiadiazine,a novel tubulin inhibitor,evokes G2/M cell cycle arrest and apoptosis in SGC-7901 and HeLa cells

Gastric cancer and cervical cancer are two major malignant tumors that threaten human health. The novel chemotherapeutic drugs are needed urgently to treat gastric cancer and cervical cancer with high anticancer activity and metabolic stability. Previously we have reported the synthesis, characterization and identification of a novel combretastatin A-4 analog, 3-(3-methoxyphenyl)-6-(3-amino-4- methoxyphenyl) -7H-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazine (XSD-7). In this study, we sought to investigate its anticancer mechanisms in a human gastric cancer cell line (SGC-7901 cells) and human cervical carcinoma cell line (HeLa cells). The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay showed that XSD-7 induced cytotoxicity in SGC-7901 and HeLa cells with inhibitory concentration 50 values of 0.11 ± 0.03 and 0.12 ± 0.05 µM, respectively. Immunofluorescence studies proved that XSD-7 inhibited microtubule polymerization during cell division in SGC-7901 and HeLa cells. Then, these cells were arrested at G2/M cell cycle and subsequently progressed into apoptosis. In further study, mitochondrial membrane potential analysis and Western blot analysis demonstrated that XSD-7 treatment-induced SGC-7901 cell apoptosis via both the mitochondria-mediated pathway and the death receptor-mediated pathway. In contrast, XSD-7 induced apoptosis in HeLa cells mainly via the mitochondria-mediated pathway. Hence, our data indicate that XSD-7 exerted antiproliferative activity by disrupting microtubule dynamics, leading to cell cycle arrest, and eventually inducing cell apoptosis. XSD-7 with novel structure has the potential to be developed for therapeutic treatment of gastric cancer and cervical cancer.