Resveratrol-mediated sensitisation to TRAIL-induced apoptosis depends on death receptor and mitochondrial signalling

Natural food products such as resveratrol have gained considerable attention as cancer chemopreventive agents. In the present study, we investigated the potential of resveratrol to overcome the resistance of tumour cells against TRAIL. While resveratrol enhanced TRAIL-induced apoptosis through G1 cell cycle arrest and survivin depletion, resveratrol failed to sensitise cells with high expression levels of Bcl-2 or FADD-DN. Interestingly, overexpression of Bcl-2 or FADD-DN did not interfere with resveratrol-mediated cell cycle arrest or survivin depletion, but blocked release of cytochrome c and Smac from mitochondria into the cytosol, enhanced caspase activation and apoptosis upon combined treatment with resveratrol and TRAIL indicating that overexpression of Bcl-2 or FADD-DN decoupled the effect of resveratrol on the cell cycle and apoptosis. Similarly, cell cycle arrest at G1 using the cell cycle specific inhibitor mimosine or downregulation of survivin expression by antisense oligonucleotides failed to enhance TRAIL-induced apoptosis in Bcl-2- or FADD-DN-transfected cells. Likewise, inhibition of caspase activity using the broad range caspase inhibitor zVAD.fmk did not interfere with resveratrol-mediated cell cycle arrest and survivin depletion, while blocking apoptosis upon combined treatment with resveratrol and TRAIL. Thus, resveratrol is a potent sensitiser for TRAIL in certain tumours. However, it may be ineffective in others, e.g. in tumours with enhanced Bcl-2 expression or defective death receptor signalling.     

Equivalent effect of DNA damage-induced apoptotic cell death or long-term cell cycle arrest on colon carcinoma cell proliferation and tumour growth

Knowledge of the type of biological reaction to chemotherapy is a prerequisite for its rational enhancement. We previously showed that irinotecan-induced DNA damage triggers in the HCT116p53(wt) colon carcinoma cell line a long-term cell cycle arrest and in HCT116p53(-/-) cells apoptosis (Magrini et al., 2002). To compare the contribution of long-term cell cycle arrest and that of apoptosis to inhibition of cell proliferation after irinotecan-induced DNA damage, we used this isogenic system as well as the cell lines LS174T (p53(wt)) and HT-29 (p53(mut)). Both p53(wt) cell lines responded to damage by undergoing a long-term tetraploid G1 arrest, whereas the p53(mut) cell lines underwent apoptosis. Cell cycle arrest as well as apoptosis caused a similar delay in cell proliferation. Irinotecan treatment also induced in mouse tumours derived from the p53(wt) cell lines a tetraploid G1 arrest and in those derived from the p53-deficient cell lines a transient G2/M arrest and apoptosis. The delay of tumour growth was in the same range in both groups, that is, arrest- and apoptosis-mediated tumour growth inhibition was comparable. In conclusion, cell cycle arrest as well as apoptosis may be equipotent mechanisms mediating the chemotherapeutic effects of irinotecan.     

Flavopiridol potentiates the cytotoxic effects of radiation in radioresistant tumor cells in which p53 is mutated or Bcl-2 is overexpressed

PURPOSE: Loss of the cell-cycle regulatory protein p53 or overexpression of the antiapoptotic protein Bcl-2 is associated with resistance to radiation in several types of cancer cells. Flavopiridol, a synthetic flavone, inhibits the growth of malignant tumors cells in vitro and in vivo through multiple mechanisms. The purpose of the present study is to clarify whether flavopiridol enhances the cytotoxic effects of radiation in tumor cells that contain dysfunction p53 or that overexpress Bcl-2. METHODS AND MATERIALS: A human glioma cell line (A172/mp53) stably transfected with a plasmid containing mutated p53 and a human cervical cancer cell line (HeLa/bcl-2) transfected with a bcl-2 expression plasmid were used. Cells were incubated with flavopiridol for 24 h after radiation, and then cell viability was determined by a colony formation assay. Foci of phosphorylated histone H2AX were also evaluated as a sensitive indicator of DNA double-strand breaks. RESULTS: Compared with the parental wild-type cells, both transfected cell lines were more resistant to radiation. Post-treatment with flavopiridol increased the cytotoxic effects of radiation in both transfected cell lines, but not in their parental wild-type cell lines. Post-treatment with flavopiridol inhibited sublethal damage repair as well as the repair of DNA double-strand breaks in response to radiation. CONCLUSIONS: Flavopiridol enhanced the cytotoxic effect of radiation in radioresistant tumor cells that harbor p53 dysfunction or Bcl-2 overexpression. A combination treatment of flavopiridol with radiation has the potential to conquer the radioresistance of malignant tumors induced by the genetic alteration of p53 or bcl-2.     

Novel mechanisms of apoptosis induced by histone deacetylase inhibitors

Histone deacetylase inhibitors (HDACIs) are a new class of chemotherapeutic drugs able to induce tumor cell apoptosis and/or cell cycle arrest; however, the molecular mechanisms underpinning their anticancer effects are poorly understood. Herein, we assessed the apoptotic pathways activated by three HDACIs, suberoylanilide hydroxamic acid, oxamflatin, and depsipeptide. We determined that all three drugs induced the accumulation of cells with a 4n DNA content and apoptosis mediated by the intrinsic apoptotic pathway. HDACI-induced mitochondrial membrane damage and apoptosis were inhibited by overexpression of Bcl-2, but not by the polycaspase inhibitor N-tert-butoxy-carbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk). Moreover, induction of a G(1)-S checkpoint through overexpression of p16(INK4A) or suppression of de novo protein synthesis also inhibited HDACI-induced cell death. Proteolytic cleavage of caspase-2, which is poorly inhibited by zVAD-fmk, was concomitant with HDACI-induced death; however, full processing of caspase-2 to the p19 active form was blocked by Bcl-2. Whereas all three drugs induce the activation of the proapoptotic Bcl-2 protein Bid upstream of mitochondrial membrane disruption, Bid cleavage in response to depsipeptide was significantly attenuated by zVAD-fmk. Suberoylanilide hydroxamic acid and oxamflatin could kill both P-glycoprotein (P-gp)(+) MDR cells and their P-gp(-) counterparts, whereas depsipeptide was shown to be a substrate for P-gp and was less effective in killing P-gp(+) cells. These data provide insight into the functional profile of three HDACIs and are important for the development of more rational approaches to chemotherapy, where information regarding the genetic profile of the tumor is matched with the functional profile of a given chemotherapeutic drug to promote favorable clinical responses.     

Overexpression of p53 protein during pancreatitis.

Overexpression of p53 correlates with neoplasia in many cytological specimens. To test the specificity of overexpressed p53 as a tumour marker for the detection of pancreatic cancer, we analysed cytological specimens of pancreatic juice samples from patients with pancreatitis or pancreatic carcinoma (n = 42) for p53 protein overexpression. p53 protein overexpression was found in 59% of patients with pancreatitis and 67% of patients with pancreatic carcinoma. Thus, the assessment of p53 protein overexpression is not useful in the diagnosis of pancreatic cancer. Overexpressed p53 during pancreatitis appears to be wild-type p53. Overexpression of p53 may result from DNA damage occurring during chronic inflammation. It is well established that p53 can induce apoptosis upon DNA damage. Consequently, we found apoptotic cell death in five out of five tested cytological preparations from patients with pancreatitis as well as in one out of one pancreatic carcinoma specimen.     

Overcoming the radioresistance of prostate cancer cells with a novel Bcl-2 inhibitor

Bcl-2 overexpression is an important mechanism underlying the aggressive behavior of prostate cancer cells and their resistance to radio- or chemotherapy. HA14-1, a recently discovered organic Bcl-2 inhibitor, potently induces apoptosis in various human cancer cells. Sequential exposure of radioresistant LNCaP (wild-type (wt) p53), LNCaP/Bcl-2 (wt p53) and PC3 (mutant p53) prostate cancer cells to a minimally cytotoxic concentration of 10 microM HA14-1 for 1 h followed by 1-6 Gy gamma radiation, resulted in a highly synergistic (combination index <1.0) induction of cell death as determined by an apoptosis assay at 72 h, and a clonogenicity assay at 12 days, after the initial treatment. The reverse treatment sequence did not cause a synergistic induction of cell death. When compared to individual treatments, cell death induced by the combined treatment was associated with dramatically increased reactive oxygen species (ROS) generation, c-Jun N-terminal kinase (JNK) activation, Bcl-2 phosphorylation, cytochrome c release, caspase-3 activation and DNA fragmentation. Exposure to either 200 microg/ml of the antioxidant alpha-tocopherol or 10 microM JNK inhibitor SP600125 before the combined treatment resulted in decreased activation of JNK and caspase-3 as well as decreased DNA fragmentation. However, treatment with the pancaspase inhibitor carbobenzoxyl-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone before the combined treatment inhibited apoptosis without affecting JNK activation, and this inhibitory effect was enhanced in the presence of alpha-tocopherol or SP600125. Taken together, our results indicate that HA14-1 potently sensitizes radioresistant LNCaP and PC3 cells to gamma radiation, regardless of the status of p53. ROS and JNK are important early signals that trigger both caspase-dependent and -independent cell death pathways and contribute to the apoptotic synergy induced by the combined treatments.     

Novel HSP90 inhibitors, NVP-AUY922 and NVP-BEP800, radiosensitise tumour cells through cell-cycle impairment, increased DNA damage and repair protraction

Background:

Heat-shock protein 90 (Hsp90) has a crucial role in both the stabilisation and regulation of various proteins, including those related to radioresistance. Inhibition of Hsp90 may therefore provide a strategy for enhancing the radiosensitivity of tumour cells. This study explores the responses of four tumour cell lines (A549, GaMG, HT 1080 and SNB19) to combined treatment with ionising radiation (IR) and two novel inhibitors of Hsp90, NVP-AUY922 and NVP-BEP800. The techniques used included cell and colony counts, expression of Hsp90, Hsp70, Akt, survivin, cleaved caspase 3, p53, cell-cycle progression and associated proteins. DNA damage was analysed by histone γH2AX and Comet assays.

Results:

We found that NVP-AUY922 and NVP-BEP800 enhanced radiosensitivity in all tested cell lines. In contrast, only two cell lines (HT 1080 and GaMG) exhibited an increased rate of apoptosis after drug pretreatment, as revealed by western blot. In all tested cell lines, the expression of histone γH2AX, a marker of DNA double-strand breaks, after combined drug-IR treatment was higher and its decay rate was slower than those after each single treatment modality. Drug-IR treatment also resulted in impaired cell-cycle progression, as indicated by S-phase depletion and G2/M arrest. In addition, the cell cycle-associated proteins, Cdk1 and Cdk4, were downregulated after Hsp90 inhibition.

Interpretation:

These findings show that the novel inhibitors of Hsp90 can radiosensitise tumour cell lines of different entities through destabilisation and depletion of several Hsp90 client proteins, thus causing the depletion of S phase and G2/M arrest, increased DNA damage and repair protraction and, to some extent, apoptosis. The results might have important implications for the radiotherapy of solid tumours.     

Chemical radiosensitizers for use in radiotherapy

Radiosensitizers are intended to enhance tumour cell killing while having much less effect on normal tissues. Some drugs target different physiological characteristics of the tumour, particularly hypoxia associated with radioresistance. Oxygen is the definitive hypoxic cell radiosensitizer, the large differential radiosensitivity of oxic vs hypoxic cells being an attractive factor. The combination of nicotinamide to reduce acute hypoxia with normobaric carbogen breathing is showing clinical promise. 'Electron-affinic' chemicals that react with DNA free radicals have the potential for universal activity to combat hypoxia-associated radioresistance; a nitroimidazole, nimorazole, is clinically effective at tolerable doses. Hypoxia-specific cytotoxins, such as tirapazamine, are valuable adjuncts to radiotherapy. Nitric oxide is a potent hypoxic cell radiosensitizer; variations in endogenous levels might have prognostic significance, and routes to deliver nitric oxide specifically to tumours are being developed. In principle, many drugs can be delivered selectively to hypoxic tumours using either reductase enzymes or radiation-produced free radicals to activate drug release from electron-affinic prodrugs. A redox-active agent based on a gadolinium chelate is being evaluated clinically. Pyrimidines substituted with bromine or iodine are incorporated into DNA and enhance free radical damage; fluoropyrimidines act by different mechanisms. A wide variety of drugs that influence the nature or repair of DNA damage are being evaluated in conjunction with radiation; it is often difficult to define the mechanisms underlying chemoradiation regimens. Drugs being evaluated include topoisomerase inhibitors (e.g. camptothecin, topotecan), and the hypoxia-activated anthraquinone AQ4N; alkylating agents include temozolomide. Drugs involved in DNA repair pathways being investigated include the potent poly(ADP ribose)polymerase inhibitor, AG14,361. Proteins involved in cell signalling, such as the Ras family, are attractive targets linked to radioresistance, as are epidermal growth factor receptors and linked kinases (drugs including vandetanib [ZD6,474], cetuximab and gefitinib), and cyclooxygenase-2 (celecoxib). The suppression of radioprotective thiols seems to offer more potential with alkylating agents than with radiotherapy, although it remains a strategy worthy of exploration.     

Growth arrest and cell death in the breast tumor cell in response to ionizing radiation and chemotherapeutic agents which induce DNA damage

Breast tumor cells are relatively refractory to apoptosis in response to modalities which induce DNA damage such as ionizing radiation and the topoisomerase II inhibitor, adriamycin. Various factors which may modulate the apoptotic response to DNA damage include the p53 status of the cell, levels and activity of the Bax and Bcl-2 families of proteins, activation of NF-kappa B, relative levels of insulin like growth factor and insulin-like growth factor binding proteins, activation of MAP kinases and PI3/Akt kinases, (the absence of) ceramide generation and the CD95 (APO1/Fas) signaling pathway. Prolonged growth arrest associated with replicative senescence may represent an alternative and reciprocal response to DNA-damage induced apoptosis that is p53 and/or p21^waf1/cip1 dependent while delayed apoptosis may occur in p53 mutant breast tumor cells which fail to maintain the growth-arrested state. Clearly, the absence of animmediate apoptotic response to DNA damage does not eliminate other avenues leading to cell death and loss of self-renewal capacity in the breast tumor cell. Nevertheless, prolonged growth arrest (even if ultimately succeeded by apoptotic or necrotic cell death) could provide an opportunity for subpopulations of breast tumor cells to recover proliferative capacity and to develop resistance to subsequent clinical intervention.     

Targeting polymerase θ impairs tumorigenesis and enhances radiosensitivity in lung adenocarcinoma

Radioresistance remains a major obstacle to efficacious radiotherapy in non–small-cell lung cancer (NSCLC). DNA replication proteins are novel targets for radiosensitizers. POLQ is a DNA polymerase involved in DNA damage response and repair. We found that POLQ is overexpressed in NSCLC and is clinically correlated with high tumor stage, poor prognosis, increased tumor mutational burden, and ALK and TP5 mutation status; POLQ inhibition impaired lung tumorigenesis. Notably, POLQ expression was higher in radioresistant lung cancer cells than in wild-type cancer cells. Moreover, POLQ expression was further increased in radioresistant cells after radiation. Enhanced radioresistance is through a prolonged G2/M phase and faster repair of DNA damage, leading to reduced radiation-induced apoptosis. Novobiocin (NVB), a POLQ inhibitor, specifically targeted cancer cells. Genetic knockdown of POLQ or pharmacological inhibition by NVB decreased radioresistance in lung adenocarcinoma while causing little toxicity to normal pulmonary epithelial cells. In conclusion, POLQ is a promising and practical cancer-specific target to impair tumorigenesis and enhance radiosensitivity in NSCLC.     

Abrogation of radioresistance in glioblastoma stem‐like cells by inhibition of ATM kinase

Resistance to radiotherapy in glioblastoma (GBM) is an important clinical problem and several authors have attributed this to a subpopulation of GBM cancer stem cells (CSCs) which may be responsible for tumour recurrence following treatment. It is hypothesised that GBM CSCs exhibit upregulated DNA damage responses and are resistant to radiation but the current literature is conflicting. We investigated radioresistance of primary GBM cells grown in stem cell conditions (CSC) compared to paired differentiated tumour cell populations and explored the radiosensitising effects of the ATM inhibitor KU‐55933.We report that GBM CSCs are radioresistant compared to paired differentiated tumour cells as measured by clonogenic assay. GBM CSC''s display upregulated phosphorylated DNA damage response proteins and enhanced activation of the G2/M checkpoint following irradiation and repair DNA double strand breaks (DSBs) more efficiently than their differentiated tumour cell counterparts following radiation.Inhibition of ATM kinase by KU‐55933 produced potent radiosensitisation of GBM CSCs (sensitiser enhancement ratios 2.6–3.5) and effectively abrogated the enhanced DSB repair proficiency observed in GBM CSCs at 24 h post irradiation. G2/M checkpoint activation was reduced but not abolished by KU‐55933 in GBM CSCs.ATM kinase inhibition overcomes radioresistance of GBM CSCs and, in combination with conventional therapy, has potential to improve outcomes for patients with GBM.     

« Dose-painting » : mythe ou réalité ?

“Dose-painting” radiotherapy allows for a heterogeneous delivery of radiation within the tumour volume by targeting radioresistant areas defined by functional imaging. Within gross tumour volume, it is possible to define one or more target volumes based on biology (biological target volume [BTV]) and to apply a strategy of intensity modulated radiation therapy (IMRT) that will deliver a higher dose to these regions. In this review of the literature, we will highlight the biological elements responsible for radioresistance, and how to image them, then we will detail the radiotherapy techniques necessary for this approach, before presenting clinical results in various situations (head and neck tumours, prostate, brain tumours, etc.). Despite many difficulties that make dose-painting IMRT unusable in routine nowadays, biology-guided radiation therapy represents one of the major pathways of development of radiotherapy in the coming years.     

Expression of Cox-2 protein in radioresistant laryngeal cancer.

BACKGROUND: Radiotherapy is the principal modality used to treat early stage laryngeal cancer. Unfortunately treatment failures occur in 10-25% of patients. Subsequent salvage surgery is technically more difficult, with increased complication and failure rates. The ability to predict or prevent radioresistance would improve the poor survival associated with this disease. Cox-2 is an inducible enzyme involved with prostaglandin synthesis. We investigated a potential role for Cox-2 in predicting radioresistance in laryngeal cancer. PATIENTS AND METHODS: Using immunohistochemical techniques we examined the expression of Cox-2 protein in 122 pre-treatment laryngeal biopsies. All tumours were treated with single modality radiotherapy (curative intent). The group comprised of 61 radioresistant and 61 radiosensitive tumours matched for T stage, laryngeal subsite, gender and smoking history. RESULTS: Cox-2 expression was detected in 41 of 61 (67%) biopsy samples from patients with radioresistant tumours and 25 of 61 (41%) radiosensitive tumours. Overexpression was significantly associated with radioresistant tumours (P = 0.004). Cox-2 has a 67% accuracy in predicting radiotherapy failure. CONCLUSION: Cox-2 may have prognostic value in predicting response to radiotherapy. Cox-2 inhibitors such as NS-398 have been shown to enhance the effects of radiotherapy. We suggest that their use may be beneficial in patients who are destined to fail radiotherapy.     

鼻咽癌放射抗拒相关microRNAs研究进展

鼻咽癌是一种起源于鼻咽部上皮组织的鳞状细胞恶性肿瘤,在东南亚和中国华南地区具有很高的发病率,目前放疗已成为鼻咽癌的首选治疗方式。放射抗拒是鼻咽癌放疗成功的主要障碍,寻找鼻咽癌放射抗拒相关的生物标志物,明确放射抗拒的产生机制,对治疗具有重大意义。MicroRNAs通过结合靶mRNA的3’UTR 从而诱导其翻译抑制或降解,调节蛋白的表达,参与放疗反应相关的所有重要的细胞过程如DNA损伤反应与修复、细胞凋亡、增殖以及血管生成的调节。近年来鼻咽癌放射抗拒相关microRNAs的研究成为热点,本文就microRNAs及其潜在的作用机制进行综述。